KR20210126029A - Compositions and methods for evading humoral immunity - Google Patents

Abstract

The present invention provides, in part, compositions and methods for transiently removing neutralizing antibodies to AAV vectors. Such compositions and methods are intended to expand a cohort of patients suitable for gene therapy and to re-dos/re-administer AAV to patients previously treated with AAV vectors.

Description

Compositions and methods for evading humoral immunity

relation Cross-reference to application

This application claims priority to U.S. Provisional Application No. 62/914,682, filed October 14, 2019, and U.S. Provisional Application No. 62/797,495, filed January 28, 2019, each of which is incorporated herein by reference in its entirety.

Electronically Submitted Documents Description of the file

The content of the document file submitted electronically with this application is hereby incorporated by reference in its entirety: a computer readable format copy of the Sequence Listing (filename: STRD_014_02WO_SeqList_ST25.txt, recorded date January 28, 2020, file size ~180 kb).

technical field

This application relates generally to the field of gene therapy, eg, gene therapy using adeno-associated virus (AAV) vectors. More specifically, the present invention relates to a composition for improving the effect of a recombinant AAV treatment and a method for improving the effect of a recombinant AAV treatment using the composition and method for lowering neutralizing antibodies to recombinant AAV.

federal funding

This invention was made with government support of Federal Grant No. R01HL089221 from the National Heart, Lung and Blood Association (NIH/NHLBI) and Federal Grant No. R01GM127708 from the National Institute of General Medical Sciences (NIH/NIGMS). The federal government has certain rights in this invention.

Adeno-associated virus (AAV) is a helper-dependent parvovirus that can be used to deliver therapeutic genes in humans. Since the first AAV1-based gene therapy was approved by regulatory agencies in 2012, encouraging results have been published in clinical gene therapy trials using recombinant AAV vectors for Levers congenital amaurosis, hemophilia and other diseases.

Although gene therapy trials use several native AAV isolates, the same exclusion criteria of low or undetectable anti-AAV neutralizing antibody (NAb) titers in future patients who wish to participate are common. These eligibility criteria were established due to the high prevalence of pre-existing anti-AAV Nabs resulting from natural exposure in the human population; For example, the overall prevalence of anti-AAV1 NAb positive at titers >1:2 in human subjects with heart failure is -60%. In addition, most patients with high Nab titers to AAV serotype 2 also had measurable titers to AAV1, suggesting cross-reactivity between serotypes. Nabs substantially reduce the gene transfer efficiency of AAV vectors through opsonization, so their clearance is accelerated, their biodistribution is altered, their binding to cell surface receptors is blocked, and/or essential for efficient transduction It will have a negative effect on the process after attachment.

Attempts to develop solution strategies for existing anti-AAV Nabs have focused on AAV capsid manipulation and attraction, transient pharmacological immunomodulation, and plasmapheresis. These approaches have been shown to have limited potential to enhance AAV gene delivery by avoiding or reducing the Nabs already present in preclinical animal models and humans.

Accordingly, in the art, in order to improve the efficiency of gene transfer using an AAV vector, compositions and methods for lowering, eliminating, or inactivating the production of antibodies against the existing anti-AAV Nab after administration of the AAV vector to an individual are provided. is being demanded

The present invention provides compositions and methods for reducing the content of neutralizing antibodies to one or more recombinant adeno-associated virus (AAV) vectors in a subject in need thereof. The compositions and methods described herein can improve gene transfer efficiency, for example, by increasing the circulation time and/or infectivity of AAV in an individual. The compositions and methods described herein also allow for re-administration of a therapeutic AAV to a subject previously treated with a therapeutic AAV. In some embodiments, a wild-type or mutant form (or fragment thereof) of an antibody-degrading enzyme, such as IdeZ, is administered to lower NAb in a subject in need thereof.

In some embodiments, the present invention provides a method of reducing the content of neutralizing antibodies to a recombinant adeno-associated virus (AAV) vector in a subject in need thereof, comprising administering to the subject an effective amount of a composition that promotes degradation of the neutralizing antibody to provide.

In addition, the present invention comprises administering to an individual an effective amount of a composition that (a) promotes the degradation of a neutralizing antibody to an AAV vector, and/or (b) reduces binding of the neutralizing antibody to an Fc receptor, Methods are provided for preparing a subject in need for treatment with a recombinant adeno-associated virus (AAV) vector.

In addition, the present invention provides the steps of: (i) (a) promoting degradation of a neutralizing antibody to an AAV vector, and/or (b) administering to an individual an effective amount of a composition that reduces binding of the neutralizing antibody to an Fc receptor; ; and (ii) administering to the individual an effective amount of the AAV vector.

The present invention also relates to (i) (a) promoting degradation of a neutralizing antibody to a first recombinant AAV vector and/or a second recombinant AAV vector, and/or (b) binding of said neutralizing antibody to an Fc receptor. administering to the subject an effective amount of a composition that lowers and (ii) administering to the individual an effective amount of a second recombinant AAV vector; .

The present invention also provides an AAV vector, and (a) promotes degradation of an antibody against an AAV vector or a recombinant protein encoded by a heterologous nucleic acid; and/or (b) a neutralizing antibody against an adeno-associated virus (AAV) vector comprising a heterologous nucleic acid in a subject in need thereof, comprising administering to the subject an effective amount of a composition that reduces binding of the antibody to an Fc receptor. provide a way to lower it.

The compositions described herein may include, for example, an antibody-degrading enzyme or fragment thereof. In some embodiments, the composition described above comprises a vector comprising a polynucleotide encoding an antibody-degrading enzyme or fragment thereof. In some embodiments, the antibody-degrading enzyme or fragment thereof has cysteine protease activity. In some embodiments, the antibody-degrading enzymes specifically cleave IgG. In some embodiments, the antibody-degrading enzymes have at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO:1. have

Hereinafter, these and other implementations will be described in more detail.

Figure 1A-1B. 1A depicts a map of the GST-IdeZ expression vector. Using this vector, recombinant GST-IdeZ was expressed in E. coli and purified using glutathione sepharose. Figure 1B shows a gel picture used to visualize the purified GST-IdeZ.
2A-2D. Figure 2A shows a photograph of a Coomassie-stained SDS-PAGE gel showing the band pattern of a recombinant IgG sample untreated (-) or treated (+) with recombinant IdeZ. FIG. 2B depicts identically stained SDS-PAGE gel photographs showing band patterns of mouse serum, primate serum and human serum samples untreated (-) or treated (+) with rIdeZ. 2A and 2B, * denotes an IgG heavy chain cleavage product (~ 31 kDa). Figure 2C depicts the results of a similar experiment demonstrating that recombinant IdeZ cleaves dog-derived serum IgG, and Figure 2D depicts IdeZ cleavage of human patient-derived serum IgG.
3A-3B . 3A is a Western blot photograph showing that IdeZ cutting vivo human IVIG in mice. * indicates cleavage products. Figure 3B is an SDS-PAGE gel picture showing coma upon staining the band pattern after a test tube handling the IdeZ human IVIG.
4 . Figure 4 is a my peritoneal the immune globulin (IVIG) human intravenously to mice after the next 24 hours PBS or recombinant IdeZ (2.5 mg / kg), and ^{AAV8-Luc (5 x 10 12} vg / kg) of the injection of the experiment A graph showing the results is presented. The level of the luciferase (Luc) transgene in the liver was analyzed 4 weeks after injection into the liver. Luciferase expression levels were normalized to total tissue protein concentration and expressed in relative luminous units per gram of liver tissue (RLU). All experiments were performed in 3 sets. LOD = limit of detection. * p < 0.05.
Figure 5 . 5 shows the crystal structure of the protein IdeZ. Each panel shows several views.
6 . 6, the process (+) or no treatment at 37 ℃ for recombinant GST-IdeZ (1 ㎍) 3 hours a given serum sample were (). The reaction was diluted 1:10 and analyzed by SDS-PAGE under reducing conditions. The gel was then stained with Coomassie Blue. * indicates IgG heavy chain cleavage product (~31 kDa).
7 . Figure 7 shows the results of experiments in mouse peritoneal injection of 8 mg human IVIG. After 24 hours, the same mouse was intravenously injected with PBS (-) or recombinant GST-IdeZ (2.5 mg/kg) (+). Blood samples were drawn before IVIG injection, and blood samples were drawn 24 hours, 48 hours and 72 hours after IVIG injection. Blood samples were analyzed by SDS-PAGE and Western blotting. IVIG was probed with HRP-conjugated goat anti-humIgG (1:10,000) secondary antibody. Each lane is a blood sample obtained from an individual mouse.
8A-8B . Figure 8A-8B shows the results of experiments in mouse peritoneal injection of 8 mg human IVIG. After 24 hours, PBS (-) ( FIG. 8A , left panel) or recombinant GST-IdeZ was administered to the same mouse at a dose of 0.25 mg/kg ( FIG. 8A , right panel), 1 mg/kg ( FIG. 8B , left panel) or Intravenous injection at 2.5 mg/kg ( FIG. 8B , right panel) (+). Blood samples were drawn 72 hours after IVIG injection and analyzed by SDS-PAGE and Western blotting. IVIG was probed with HRP-conjugated goat anti-humIgG (1:10,000) secondary antibody. Each lane is a blood sample obtained from an individual mouse.
Figure 9 . Figure 9 shows the results of experiments in mouse peritoneal injection of 8 mg human IVIG. After 24 hours, PBS (-) or recombinant GST-IdeZ (1 mg/kg) (+) was intravenously injected into the same mouse. Blood samples were drawn 72 hours after IVIG injection and analyzed by SDS-PAGE and Western blotting. IVIG was probed with HRP-conjugated goat anti-humIgG (1:10,000) secondary antibody or HRP-conjugated goat anti-humIgG Fc (1:10,000) secondary antibody.
Figure 10 . Figure 10 shows the neutralization profile of AAV8-Luc using human IVIG. Human IVIG samples were treated with or without GST-IdeZ (1 μg) and serially diluted in 2-fold increments from 1:1000 to 1:102,400. The samples were then co-cultured with AAV8-Luc (100,000 vg/cell) in vitro. The solid line shows the relative transduction efficiency of IVIG-treated AAV8-Luc and IVIG-treated AAV8-Luc pre-incubated with GST-IdeZ at various IVIG dilutions. Error bars represent SEM (n=3).
11 . Figure 11 shows the number of AAV8-Luc copies in mouse liver. Each bar is a different mouse. The first 8 bars are mice injected with AAV8 alone (PBS-PBS-AAV8). The next 6 bars are mice injected with recombinant IdeZ and AAV8-Luc (PBS-IdeZ-AAV8). The next 6 bars are mice injected with IVIG followed by AAV8-Luc (IVIG-PBS-AAV8). The last 8 bars are mice injected with both IdeZ and AAV8-Luc after IVIG injection (IVIG-IdeZ-AA8). The number of copies of the vector genome per cell was calculated.
12A-12D. Figure 12A-12D is a graph showing the results of experiments the injection (2.5 mg / kg) PBS or recombinant GST-IdeZ my mouse peritoneal injection of 8 mg human IVIG and then after 72 hours is presented. At 72 hours after IdeZ treatment, AAV9-Luc (2 x 10 ¹¹ vg/kg) was intravenously injected into mice. Hepatic luciferase (Luc) transgene expression levels were analyzed 4 weeks after injection in liver and heart. Luciferase expression levels were normalized to total tissue protein concentration and expressed as relative luminous units per gram of liver tissue (RLU). All experiments were performed in 3 sets. LOD = limit of detection.
13A-13B. 13A-13B show % transduction in liver ( FIG. 13A ) and heart ( FIG. 13B ). Serum samples were obtained from 18 human patients. 100 μl of each human patient serum sample was injected intraperitoneally into two mice. After that, after 72 hours, PBS or recombinant GST-IdeZ (2.5 mg/kg) was intravenously injected into the mice. After 72 hours of IdeZ treatment, AAV9-Luc (2 x 10 ¹¹ vg/mouse) was injected intravenously into mice. Levels of transduction in liver and heart were analyzed 4 weeks post-injection. Transduction levels were normalized to control mice injected with AAV9-Luc (2×10 ^{11 vg/mouse) without serum treatment.}

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the detailed description herein is for the purpose of describing specific embodiments only and is not intended to be limiting.

All publications, patent applications, patents, GenBank or other accession numbers and other references mentioned herein are incorporated herein by reference in their entirety.

All amino acid positions in the AAV capsid protein herein and in the appended claims are indicated according to the VP1 capsid subunit numbering system. Those skilled in the art will appreciate that the modifications mentioned herein can result in modifications to the VP1, VP2 and/or VP3 capsid subunits if inserted into the AAV cap gene. Alternatively, the capsid subunits can be independently expressed such that modification is achieved in only one or two of the capsid subunits (VP1, VP2, VP3, VP1 + VP2, VP1 + VP3, or VP2 + VP3).

Unless the context dictates otherwise, it is expressly intended that the various features described herein may be used in any combination.

Justice

The following terms are used in the description herein and in the appended claims:

The singular forms (“a,” “an” and “the”) are intended to encompass the plural forms as well, unless the context clearly dictates otherwise.

Also, as used herein, the term "about" when referring to a measurable value, such as the length, amount, time, temperature, etc. of a polynucleotide or polypeptide sequence, is ± 20%, ± 10%, ± 5 of the specified value. %, ±1%, ±0.5% or even ±0.1%.

Also, as used herein, "and/or" means any and all possible combinations of one or more of the linked listed items, as well as instances other than combinations when construed as a selection ("or"), encompassing all do.

Reference to numerical ranges herein, unless otherwise indicated herein, is primarily intended to be used as a shorthand way of referring to each individual value falling within that range, each individual value being incorporated into the specification as if individually recited herein. do. For example, if a concentration range is stated as 1% to 50%, then numerical values such as 2% to 40%, 10% to 30%, or 1% to 3%, etc. are intended to be expressly recited herein. These are specifically intended examples only, and all possible combinations of numerical values therebetween, including the recited minimum and maximum values, are to be considered as expressly recited herein.

As used herein, the term "adeno-associated virus" (AAV) refers to AAV type 1, AAV type 2, AAV type 3 (including types 3A and 3B), AAV type 4, AAV type 5, AAV type 6, AAV type 7, AAV type 8, AAV type 9, AAV type 10, AAV type 11, AAV type 12, AAV type 13, AAV type rh32.33, AAV type rh8, AAV type rh10, AAV type rh74, AAV type hu.68, avian AAV , Bovine AAV, Dog AAV, Horse AAV, Sheep AAV, Snake AAV, Bearded Dragon AAV, AAV2i8, AAV2g9, AAV-LK03, AAV7m8, AAV Anc80, AAV PHP.B and other AAVs currently known or to be discovered in the future These include, but are not limited to. See, for example, BERNARD N. FIELDS et al., VIROLOGY, volume 2, chapter 69 (4th ed., Lippincott-Raven Publishers). A number of AAV serotypes and clades have been identified (eg, Gao et al, (2004) J. Virology 78:6381-6388; Moris et al, (2004) Virology 33-:375-383; and Tables). see 2 ). In some embodiments, the AAV vector is selected from any of the AAV vectors described in Table 1 of WO 2019/028306, which is incorporated herein by reference in its entirety.

As used herein, the term “chimeric AAV” refers to an AAV comprising a capsid protein having regions, domains, and individual amino acids derived from two or more different AAV serotypes. In some embodiments, the chimeric AAV comprises a capsid protein consisting of a first region derived from a first AAV serotype and a second region derived from a second serotype. In some embodiments, the chimeric AAV comprises a capsid protein consisting of a first region derived from a first AAV serotype, a second region derived from a second serotype and a third region derived from a third serotype. In some embodiments, a chimeric AAV may comprise regions, domains, individual amino acids derived from two or more of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, and/or AAV12. have. For example, a chimeric AAV may comprise regions, domains and/or individual amino acids derived from a first AAV serotype and a second AAV serotype shown below (Table 1), wherein AAVX+Y is from AAVX and AAVY. It is a chimeric AAV containing the derived sequence.

By including individual amino acids or regions derived from different AAV serotypes in one capsid protein, it is possible to obtain capsid proteins with a plurality of desired properties, each derived from a plurality of AAV serotypes.

The genomic sequences of the various serotypes of AAV and the genomic sequences of autonomous parvoviruses as well as sequences of native terminal repeats (TRs), Rep proteins and capsid subunits are known in the art. These sequences can be found in the literature or in public databases such as GenBank. For example, GenBank accession numbers NC_002077, NC_001401, NC_001729, NC_001863, NC_001829, NC_001862, NC_000883, NC_001701, NC_001510, NC_006152, NC_006261, AF063497, U897902, AF043301, J028702 AF043301, J028702 AF043301, J028702 AF043301 , AY028226, AY028223, NC_001358, NC_001540, AF513851, AF513852, AY530579; These contents are incorporated herein by reference for parvovirus and AAV nucleic acid and amino acid sequences. See, eg, Srivistava et al., (1983) J. Virology 45:555; Chiorini et al, (1998) J Virology 71:6823; Chiorini et al., (1999) J. Virology 73: 1309; Bantel-Schaal et al., (1999) J Virology 73:939; Xiao et al, (1999) J Virology 73:3994; Muramatsu et al., (1996) Virology 221:208; Shade et al, (1986) J. Virol. 58:921; Gao et al, (2002) Proc. Nat. Acad. Sci. USA 99:11854; Moris et al, (2004) Virology 33:375-383; International Patent Publication Nos. WO 00/28061, WO 99/61601, WO 98/11244; and US Patent No. 6,156,303; These contents are incorporated herein by reference for parvovirus and AAV nucleic acid and amino acid sequences. See also Table 2 . The capsid structures of autonomous parvoviruses and AAVs are described in more detail in BERNARD N. FIELDS et al., VIROLOGY, volume 2, chapters 69 & 70 (4th ed., Lippincott-Raven Publishers). In addition, AAV2 (Xie et al., (2002) Proc. Nat. Acad. Sci. 99: 10405-10), AAV9 (DiMattia et al., (2012) J. Virol. 86:6947-6958), AAV8 ( Nam et al, (2007) J. Virol. 81: 12260-12271), AAV6 (Ng et al., (2010) J. Virol. 84:12945-12957), AAV5 (Govindasamy et al. (2013) J. Virol. 87, 11187-11199), AAV4 (Govindasamy et al. (2006) J. Virol. 80:11556-11570), AAV3B (Lerch et al., (2010) Virology 403:26-36), BPV (Kailasan) et al., (2015) J. Virol. 89:2603-2614) and CPV (Xie et al, (1996) J. Mol. Biol. 6:497-520 and Tsao et al, (1991) Science 251:1456) -64), refer to the crystal structure.

GenBank registration number GenBank registration number GenBank registration number whole genome Clade C Rh57 AY530569 Adeno-associated virus 1 NC_002077, AF063497 Hu9 AY530629 Rh50 AY530563 adeno-associated virus 2 NC_001401 Hu10 AY530576 Rh49 AY530562 adeno-associated virus 3 NC_001729 Hu11 AY530577 Hu39 AY530601 Adeno-associated virus 3B NC_001863 Hu53 AY530615 Rh58 AY530570 adeno-associated virus 4 NC_001829 Hu55 AY530617 Rh61 AY530572 Adeno-associated virus 5 Y18065, AF085716 Hu54 AY530616 Rh52 AY530565 adeno-associated virus 6 NC_001862 Hu7 AY530628 Rh53 AY530566 Bird AAV ATCC VR-865 AY186198, AY629583, NC_004828 Hu18 AY530583 Rh51 AY530564 Avian AAV strain DA-1 NC_006263, AY629583 Hu15 AY530580 Rh64 AY530574 Bobine AAV NC_005889, AY388617, AAR26465 Hu16 AY530581 Rh43 AY530560 AAV11 AAT46339, AY631966 Hu25 AY530591 AAV8 AF513852 AAV12 ABI16639, DQ813647 Hu60 AY530622 Rh8 AY242997 Clade A Ch5 AY243021 Rh1 AY530556 AAV1 NC_002077, AF063497 Hu3 AY530595 Clade F AAV6 NC_001862 Hu1 AY530575 Hu14 (AAV9) AY530579 Hu.48 AY530611 Hu4 AY530602 Hu31 AY530596 Hu 43 AY530606 Hu2 AY530585 Hu32 AY530597 Hu 44 AY530607 Hu61 AY530623 HSC1 MI332400.1 Hu 46 AY530609 Clade D HSC2 MI332401.1 Clade B Rh62 AY530573 HSC3 MI332402.1 Hu. 19 AY530584 Rh48 AY530561 HSC4 MI332403.1 Hu. 20 AY530586 Rh54 AY530567 HSC5 MI332405.1 Hu 23 AY530589 Rh55 AY530568 HSC6 MI332404.1 Hu22 AY530588 Cy2 AY243020 HSC7 MI332407.1 Hu24 AY530590 AAV7 AF513851 HSC8 MI332408.1 Hu21 AY530587 Rh35 AY243000 HSC9 MI332409.1 Hu27 AY530592 Rh37 AY242998 HSC11 MI332406.1 Hu28 AY530593 Rh36 AY242999 HSC12 MI332410.1 Hu 29 AY530594 Cy6 AY243016 HSC13 MI332411.1 Hu63 AY530624 Cy4 AY243018 HSC14 MI332412.1 Hu64 AY530625 Cy3 AY243019 HSC15 MI332413.1 Hu13 AY530578 Cy5 AY243017 HSC16 MI332414.1 Hu56 AY530618 Rh13 AY243013 HSC17 MI332415.1 Hu57 AY530619 Clade E Hu68 Hu49 AY530612 Rh38 AY530558 Clone Isolation Hu58 AY530620 Hu66 AY530626 AAV5 Y18065, AF085716 Hu34 AY530598 Hu42 AY530605 AAV 3 NC_001729 Hu35 AY530599 Hu67 AY530627 AAV 3B NC_001863 AAV2 NC_001401 Hu40 AY530603 AAV4 NC_001829 Hu45 AY530608 Hu41 AY530604 Rh34 AY243001 Hu47 AY530610 Hu37 AY530600 Rh33 AY243002 Hu51 AY530613 Rh40 AY530559 Rh32 AY243003 Hu52 AY530614 Rh2 AY243007 etc Hu T41 AY695378 Bb1 AY243023 Rh74 Hu S17 AY695376 Bb2 AY243022 Bearded Dragon AAV Hu T88 AY695375 Rh10 AY243015 Snake AAV NC_006148.1 Hu T71 AY695374 Hu17 AY530582 Hu T70 AY695373 Hu6 AY530621 Hu T40 AY695372 Rh25 AY530557 Hu T32 AY695371 Pi2 AY530554 Hu T17 AY695370 Pi1 AY530553 Hu LG15 AY695377 Pi3 AY530555

As used herein, the term "tropism" means that the virus preferentially enters a particular cell or tissue, and optionally thereafter in the cell sequence(s) carried by the viral genome are expressed (eg, transcriptionally, selectively translated into ), for example, in the case of a recombinant virus, it means that heterologous target nucleic acid(s) are expressed.

One of ordinary skill in the art will appreciate that transcription of a heterologous nucleic acid sequence from the viral genome cannot be initiated without a trans-acting factor or other regulated nucleic acid sequence, for example in the case of an inducible promoter. In the case of the rAAV genome, expression of genes from the viral genome can be from a stably integrated provirus, from an unintegrated episome, as well as from any other form capable of uptake of the virus into the cell.

As used herein, "systemic tropism" and "systemic transduction" (and similar terms) mean that the viral capsid or viral vector herein is, respectively, directed to tissues throughout the body (e.g., brain, lung, skeletal muscle). , heart, liver, kidney and/or pancreas). In embodiments, systemic transduction into muscle tissue (eg, skeletal muscle, diaphragm and myocardium) is observed. In other embodiments, systemic transduction into skeletal muscle tissue is achieved. For example, in certain embodiments, essentially all skeletal muscles of the body are transduced (transduction efficiency may vary depending on the type of muscle). In certain embodiments, systemic transduction into extremity muscle, myocardium and diaphragm muscle is achieved. Optionally, the viral capsid or viral vector is administered via a systemic route (eg, a systemic route such as intravenous, intraarticular or intralymphatic). Alternatively, in other embodiments, the capsid or viral vector is delivered locally (eg, plantarly, intramuscularly, intradermally, subcutaneously, topically).

Unless otherwise indicated, "efficient transduction" or "effective tropism" or similar terms can be determined with reference to an appropriate control (e.g., at least about 50%, at least about 60%, for each transduction or tropism of the control, at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95% or more). In some embodiments, the viral vector is skeletal muscle, myocardium, diaphragm muscle, pancreas (beta-islet cells, etc.), spleen, gastrointestinal tract (eg, epithelial and/or smooth muscle), central nervous system cells, lung, articular cells and/or kidneys. is efficiently transduced or has an efficient tropism for Appropriate controls will depend on a variety of factors, including the desired tropism profile. For example, AAV8 and AAV9 are very effective in transduction of skeletal muscle, myocardium and diaphragm muscle, but transduction into liver is highly efficient, but there are problems. Thus, it is possible to identify viral vectors that efficiently transduce AAV8 or AAV9 into skeletal muscle, myocardium and/or diaphragm muscle, but with much lower liver transduction efficiency. In addition, it will be understood that the target tropism profile may indicate tropism for a plurality of target tissues, and thus an appropriate vector may be balanced. As an explanation for this, the viral vectors of the present invention may be less efficient than AAV8 or AAV9 for transduction of skeletal, myocardial and/or diaphragmatic muscles, but may nevertheless be very suitable due to their low liver transduction properties.

Likewise, it can be determined whether a virus is described as "not efficiently transduced" or "not having efficient tropism" or similar expression against the target tissue with reference to an appropriate control. In certain embodiments, the viral vector does not efficiently transduce (ie, do not have efficient tropism) into liver, kidney, gonads and/or germ cells. In certain embodiments, transduction of the inappropriate tissue(s) (eg, liver) is about 20 of the level of transduction in the desired target tissue(s) (eg, skeletal muscle, diaphragm muscle, myocardial and/or central nervous system cells). %, less than about 10%, less than about 5%, less than about 1%, less than about 0.1%.

As used herein, the term “polypeptide” encompasses both peptides and proteins, unless otherwise indicated.

A "polynucleotide" is a sequence of nucleotide bases, which can be RNA (including both naturally occurring and non-naturally occurring nucleotides), DNA, or a DNA-RNA hybrid, but in exemplary embodiments, it is single-stranded or double-stranded. DNA sequence.

As used herein, an "isolated" polynucleotide (e.g., "isolated DNA" or "isolated RNA") is from at least some other component of a naturally occurring organism or virus, e.g., a cellular or viral structural component or It refers to a polynucleotide that is separated at least in part from other polypeptides or nucleic acids that are usually found in combination with a polynucleotide. In an exemplary embodiment, an “isolated” nucleotide is enriched at least about 10-fold, about 100-fold, about 1000-fold, about 10,000-fold or more as compared to the starting material.

Likewise, an “isolated” polypeptide is defined as at least a portion from at least some other component of a naturally occurring organism or virus, for example, from a cellular or viral structural component or from other polypeptides or nucleic acids normally found in combination with the polypeptide. refers to an isolated polypeptide. In an exemplary embodiment, the "isolated" polypeptide is enriched to a level of at least about 10-fold, about 100-fold, about 1000-fold, about 10,000-fold or more compared to the starting material.

As used herein, "isolating" or "purifying" (or grammatically synonymous) a polypeptide or viral vector means to separate the polypeptide or viral vector from at least some other material contained in the starting material at least in part do. In an exemplary embodiment, an "isolated" or "purified" polypeptide or viral vector is enriched at least about 10-fold, about 100-fold, about 1000-fold, about 10,000-fold or more as compared to the starting material.

The compositions and methods disclosed herein are useful in both veterinary and medical applications. Suitable individuals include both birds and mammals. As used herein, the term “bird” includes, but is not limited to, chickens, ducks, geese, quails, turkeys, pheasants, parrots, parakeets, and the like. As used herein, the term “mammal” includes, but is not limited to, humans, non-human primates, cattle, sheep, goats, horses, cats, dogs, lemurs, and the like. Human subjects include newborn, infant, juvenile, adult and geriatric subjects. In some embodiments, the human subject may be less than 6 months old, less than 2 years old, less than 5 years old, less than 10 years old, 10-18 years old, 19-29 years old, 30-35 years old, 36-40 years old, or at least 40 years old. In an exemplary embodiment, the subject is “in need of” a method described herein. The terms “subject” and “patient” are used herein interchangeably with respect to a human subject.

A "therapeutic polypeptide" is a polypeptide that alleviates, alleviates, prevents, delays and/or stabilizes symptoms resulting from a deficiency or deficiency of a protein in a subject or cell, and/or a benefit to the subject, such as an anti-cancer effect or A polypeptide that confers improved graft survival.

The terms “treat”, “treating” or “treatment of” (and grammatical variants) refer to reducing, at least partially ameliorating, or stabilizing a subject's condition, and/or reducing the severity of one or more clinical symptoms. means that some alleviation, alleviation, reduction or stabilization is achieved, and/or the progression of the disease or disorder is delayed.

The terms "prevent", "preventing" and "prevention" (and grammatical variations) refer to preventing and/or delaying the onset of a disease, disorder and/or clinical symptom(s) in a subject, and/or of the present invention. means a reduction in the severity of the onset of a disease, disorder and/or clinical symptom(s) as compared to not practicing the method. Prevention may be complete, eg, the disease, disorder and/or clinical symptom(s) not completely developing. In addition, prevention may be partial, wherein the occurrence and/or severity of the onset and/or onset of the disease, disorder, and/or clinical symptom(s) in an individual is lower than if the method of the present invention was not practiced.

"Effective amount" means an amount sufficient to affect or ameliorate one or more symptoms of a disease, disorder or condition when administered to a subject to treat a disease, disorder or condition. An “effective amount” depends on, for example, the disease, disorder or condition and/or symptoms thereof, the severity of the disease, disorder, condition and/or symptoms thereof, the age, weight and/or health condition of the individual, and the judgment of the attending physician. may vary. Appropriate amounts in any given case will be known to those skilled in the art, or may be determined through routine experimentation. In some embodiments, the effective amount is a therapeutically effective amount.

As used herein, the term "viral vector" or "gene transfer vector" serves as a nucleic acid delivery vehicle; Refers to a viral (eg, AAC) particle comprising a vector genome (eg, viral DNA [vDNA]) packed within a virion. Alternatively, in some contexts, the term “viral vector” may be used to refer to the viral vector genome/vDNA alone.

An “rAAV vector genome” or “rAAV genome” is an AAV genome (ie, vDNA) comprising one or more heterologous nucleic acid sequences. rAAV vectors generally require only the terminal repeat(s) (TR(s)) in cis to construct the virus. All other viral sequences may not be required and may be provided in trans. Typically, the rAAV vector genome will contain only one or more TF sequences to maximize the size of the transgene that can be efficiently packed by the vector. Structural and non-structural protein coding sequences can be provided in trans (eg, from a vector such as a plasmid, or by stably incorporating the sequence into a packing cell). In embodiments, the rAAV vector genome comprises one or more TR sequences (eg, AAV TR sequences), optionally two TRs (eg, two AAV TRs), which are typically 5' of the vector genome. At the terminus and at the 3' end, the heterologous nucleic acid may be flanked, but not necessarily contiguous. The TRs may be the same or different from each other.

The term "terminal repeat" or "TR" refers to any viral terminal repeat, or any desired function such as forming a hairpin structure and functioning as a reverse terminal repeat (i.e., replication, viral packing, merging and/or provirus recovery, etc.) mediates) synthetic sequences. The TR may be an AAV TR or a non-AAV TR. For example, the TR of another parvovirus (eg, canine parvovirus (CPV), mouse parvovirus (MVM), human parvovirus B-19) or any other suitable viral sequence (eg, SU40) Non-AAV TR sequences such as the SV40 hairpin, which serve as origins of replication) can be used as TRs, which can be further modified by truncations, substitutions, deletions, insertions and/or additions. Furthermore, TR may be partially synthesized or fully synthesized.

"AAV terminal repeat" or "AAV TR" refers to serotypes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or any currently known or to be discovered in the future. It can be derived from any AAV, including, but not limited to, other AAVs (see, eg, Table 2 ). An AAV terminal repeat does not have to have a native AAV TR sequence (e.g., a native AAV TR sequence, so long as the terminal repeat mediates a desired function, such as replication, viral packing, merging and/or provirus recovery, etc.) the AAV TR sequence may be modified by insertions, deletions, truncations and/or missense mutations).

A viral vector herein may additionally be a “targeted” viral vector (eg, with directed tropism) and/or a “hybrid” parvovirus (ie, the viral TR and viral capsid are derived from several parvoviruses). can The viral vector herein may also be a duplex parvovirus particle. That is, in some embodiments, a double stranded (duplex) genome may be packed within a viral capsid herein. In addition, viral capsids or genomic elements may contain other modifications, including insertions, deletions and/or substitutions.

As used herein, the term “amino acid” encompasses all naturally occurring amino acids, modified forms thereof and synthetic amino acids.

The naturally occurring, levorotatory (L-) amino acids are shown in Table 3.

Table: Amino Acids residue and abbreviations.

amino acid residue Abbreviation 3-character code single letter code alanine Ala A arginine Arg R asparagine Asn N Aspartic Acid (Aspartate) Asp D cysteine Cys C glutamine Gln Q Glutamic Acid (Glutamate) Glu E glycine Gly G histidine His H isoleucine Ile I leucine Leu L lysine Lys K methionine Met M phenylalanine Phe F proline Pro P serine Ser S threonine Thr T tryptophan Trp W tyrosine Tyr Y valine Val V

Alternatively, the amino acid may be a modified amino acid residue (non-limiting examples of which are shown in Table 4 ), and/or post-translational modifications (eg, acetylation, amidation, formylation, hydroxylation, methylation, phosphorylation or sulfation).

Table 4: Modified Amino Acids residue

modified amino acids residue Abbreviation amino acid residue derivatives 2-aminoadipic acid Aad 3-Aminoadipic acid bAad β-alanine, β-aminopropionic acid bAla 2-Aminobutyric acid Abu 4-Aminobutyric acid, piperidic acid 4Abu 6-aminocaproic acid Acp 2-Aminoheptanoic acid Ahe 2-aminoisobutyric acid Aib 3-Aminoisobutyric acid bAib 2-Aminopimelic acid apm t-Butylalanine t-BuA Citrulline cit cyclohexylalanine Cha 2,4-diaminobutyric acid Dbu Desmosin Des 2,21-diaminopimelic acid Dpm 2,3-diaminopropionic acid Dpr N-ethylglycine EtGly N-ethylasparagine EtAsn Homoarginine hArg homocysteine hCys homoserine hSer hydroxylysine Hyl allo-hydroxylysine aHyl 3-hydroxyproline 3Hyp 4-hydroxyproline 4Hyp isodesmosine Ide Allo-Isoleucine aile Methionine sulfoxide MSO N-methylglycine, sarcosine MeGly N-methyl isoleucine Meile 6-N-methyllysine Melys N-methylvaline MeVal 2-naphthylalanine 2-Nal norvaline Nva norleucine Nle ornithine Orn 4-chlorophenylalanine Phe(4-C1) 2-fluorophenylalanine Phe(2-F) 3-fluorophenylalanine Phe(3-F) 4-fluorophenylalanine Phe(4-F) Phenylglycine Phg β-2-thienylalanine Thi

Furthermore, a non-naturally occurring amino acid may be a "non-natural" amino acid (as described in Wang et al., Annu Rev Biophys Biomol Struct. 35:225-49 (2006)). These non-natural amino acids can be usefully used to chemically link a molecule of interest to an AAV capsid protein.

As used herein, the term “domain” is intended to encompass portions of protein sequences and structures that evolve, function, and exist independently of the rest of the protein chain. Domains can form compressed three-dimensional structures, often independently stable and foldable. A domain can appear in a variety of evolutionarily related proteins. Domains vary in length from about 25 amino acids up to about 500 amino acids. A “domain” may also encompass a domain having an amino acid residue or residues substituted by conservative substitutions, derived from a wild-type protein. Since these domains are self-stable in the protein environment, domains can be genetically substituted between one protein and another to create a chimeric protein.

As used herein, the terms "mutation", "mutations", "variant" or "variants" refer to, in the native protein or AAV, one or more amino acids of the parent protein or AAV have been substituted by another amino acid and/or the parent AAV to indicate that one or more amino acids of the protein have been deleted, and/or that one or more amino acids have been inserted into the protein or AAV, and/or that one or more amino acids have been added to the parent protein or AAV. Such additions can be made either at the N-terminus or at the C-terminus of the parent protein, or both, but also internally. In some embodiments, the amino acid sequence of the variant is at least 40%, at least 50%, at least 60% or at least 70% identical to the amino acid sequence of the original protein.

As used herein, the term “vector” refers to any nucleic acid material capable of amplification in a host cell. That is, the vector may be a vector that replicates autonomously, that is, a vector, eg, a plasmid, that exists as an extrachromosomal material and whose replication is independent of chromosomal replication. Alternatively, the vector may be one that, when introduced into the host cell, is integrated into the genome of the host cell, such that it replicates along with the integrated chromosome(s). The choice of vector will often depend on the host cell into which it will be introduced. Vectors include, but are not limited to, plasmid vectors, phage vectors, viral or cosmid vectors. Vectors generally contain an origin of replication and one or more selectable genes, ie, genes whose presence encodes a product essential for cell growth or readily detectable.

The term "gene therapy" refers to a method of causing a change in the expression of an endogenous gene by exogenously administering the gene. As used herein, "gene therapy" also means replacing a defective gene encoding a defective protein or replacing an omitted gene by introducing a defective gene or a functional gene corresponding to the omitted gene into a somatic cell or stem cell of an individual in need do. Gene therapy can be accomplished by ex vivo methods, in which differentiated or somatic stem cells are harvested from the body of an individual and then normal copies of the defective gene are introduced into the explanted cells using a viral vector as a gene delivery vehicle. In addition, in vivo direct gene delivery technologies enable the delivery of genes in situ into a subject's cells using a wide range of viral vectors, liposomes, protein DNA complexes, or exposed DNA to achieve therapeutic outcomes. Also, the term "gene therapy" refers to a defect encoding a defective protein by introducing into a somatic or stem cell of an individual in need a polynucleotide that functions substantially the same as it would function if the defective gene or protein was not defective. It means replacing a gene.

The term "gene editing" refers to a DNA insertion, deletion or substitution at a specific site in the genome of an organism or cell. Gene editing can be performed using one or more targeted nuclease systems, such as the CRISPR/Cas system, the CRISPR/Cpf1 system, Zn finger nucleases, TALENs, homing endonucleases, and the like.

As used herein, the term “Fc receptor” refers to an Fcγ immunoglobulin receptor (FcγR) present on a cell. In humans, FcγR refers to one, some or all of those belonging to the receptor family, including FcγRI (CD64), FcγRIIA (CD32A), FcγRIIB (CD32B), FcγRIIIA (CD16a) and FcγRIIIB (CD16b). As used herein, the term FcyR includes naturally occurring polymorphs of FcyRI (CD64), FcyyRIIA (CD32A), FcyRIIB (CD32B), FcyRIIIA (CD16a) and FcyRIIIB (CD16b).

As described herein, cysteine proteases are enzymes that degrade proteins. Cysteine proteases generally have a conventional catalytic mechanism involving the participation of nucleophilic cysteine thiols in either the catalytic three or two molecules. In some embodiments, the cysteine protease is an IgG cysteine protease that cleaves IgG, separating the antigen binding domain (Fab) and constant domain (Fc) from each other.

neutralizing antibodies to biologics composition to reduce

Provided herein are compositions capable of lowering neutralizing antibodies to a recombinant biologic or drug substance in a subject. In some embodiments, the recombinant biologic comprises a vector comprising a heterologous nucleic acid encoding one or more recombinant proteins. In some embodiments, the vector is a recombinant viral vector, such as a recombinant AAV vector.

In some embodiments, the composition promotes clearance or degradation of the antibody to the recombinant biologic or drug substance; and/or by reducing binding of the antibody to the recombinant biologic or drug substance to Fc receptors, thereby lowering neutralizing antibodies to the recombinant biologic or drug substance in the subject. In some embodiments, the composition promotes clearance or degradation of an antibody to an AAV vector or an antibody to one or more recombinant proteins encoded by a heterologous nucleic acid; and/or by lowering the binding of the antibody to the AAV vector to the Fc receptor, thereby lowering the neutralizing antibody to the AAV vector comprising the heterologous nucleic acid in the subject.

In some embodiments, the composition is capable of degrading an adeno-associated virus (AAV) capsid protein or antibody that recognizes a virion; or an antibody-degrading enzyme or fragment thereof capable of preventing neutralization of the recombinant AAV vector. In some embodiments, the composition comprises a vector comprising a polynucleotide encoding an antibody-degrading enzyme or fragment thereof. In some embodiments, the antibody comprises IgG, IgM, IgE and/or IgA (including IgG1, IgG2a, IgG2b and/or IgG3). In an exemplary embodiment, the antibody comprises an IgG. Accordingly, in some embodiments, the composition comprises an IgG-degrading enzyme or fragment thereof. In some embodiments, the IgG-degrading enzyme or fragment thereof has cysteine protease activity.

In some embodiments, the IgG-degrading enzyme or fragment thereof is isolated or derived from a bacterium, such as a bacterium of the genus Streptococcus. In some embodiments, the IgG-degrading enzyme is at least about 50% (e.g., about 55%, about 60%, including all numbers and subranges therebetween, to the amino acid sequence of Streptococcus equi shown in SEQ ID NO: 1 below). %, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, about 99.5% or 100%) identical amino acid sequence:

MKTIAYPNKPHSLSAGLLTAIAIFSLASSNITYADDYQRNATEAYAKEVPHQITSVWSKGVTPLTPEQFRYNNEDVIHAPYLAHQGWYDITKAFDGKDNLLCGAATAGNMLHWWFDQNKTEIEAYLSKHPEKQKIIFNNQELFDLKAAIDTKDSQTNSQLFNYFRDKAFPNLSARQLGVMPDLVLDMFINGYYLNVFKTQSTDVNRPYQDKDKRGGIFDAVFTRGDQTTLLTARHDLKNKGLNDISTIIKQELTEGRALALSHTYANVSISHVINLWGADFNAEGNLEAIYVTDSDANASIGMKKYFVGINAHGHVAISAKKIEGENIGAQVLGLFTLSSGKDIWQKLS.

The sequence of SEQ ID NO: 1 corresponds to the IdeZ protein. An exemplary crystal structure for the IdeZ protein is shown in Figure 5 with views from different directions in each panel.

The IdeZ protein referred to herein is a cysteine protease identified in group A screptococci, which cleaves IgG at the lower hinge region of the heavy chain _{to dissociate one F(ab′) 2 and} one homodimeric Fc fragment to release IgG antibodies inactivate This IgG-degrading enzyme has a short half-life, is mostly cleared from the circulation rapidly, and is highly effective, but IgG clearance is transient.

In some embodiments, the IgG-degrading enzyme or fragment thereof is isolated or derived from S. pyogenes. In some embodiments, the IgG-degrading enzyme is at least about 50% of SEQ ID NO: 13 or SEQ ID NO: 14 (e.g., about 55%, about 60%, about 65%, including all numbers and subranges therebetween; about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98 %, about 99%, about 99.5% or 100%) identical amino acid sequences.

In some embodiments, the IgG-degrading enzyme or fragment thereof is a synthetase. In some embodiments, the IgG-degrading enzyme is at least about 50% with any one of SEQ ID NOs: 15-52 (e.g., about 55%, about 60%, about 65%, including all numbers and subranges therebetween) , about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, about 99.5% or 100%) identical sequences.

Compositions for lowering neutralizing antibodies described herein include antibody-degrading enzymes or fragments thereof; and a fusion protein comprising a second protein. In some embodiments, the second protein is an IgG protease.

In some embodiments, the composition for lowering neutralizing antibodies reduces the binding of antibodies to AAV vectors to Fc receptors. In some embodiments, the composition promotes rapid clearance of an AAV vector antibody by binding to the Fc receptor for that antibody. For example, the composition may promote rapid clearance of IgG to AAV vectors by binding to a receptor for IgG (eg, FcRN), and thus may promote internalization and degradation of the IgG receptor. In some embodiments, the composition comprises a therapeutic antibody. In some embodiments, the therapeutic antibody is an IgG. In some embodiments, the therapeutic antibody is rozanolixizumab. In some embodiments, the dosage of the therapeutic antibody is from 0.05 mg/kg to about 150 mg/kg, e.g., about 0.1 mg/kg, about 0.5 mg, including all numbers and subranges therebetween. /kg, about 1 mg/kg, about 2 mg/kg, about 3 mg/kg, about 4 mg/kg, about 5 mg/kg, about 6 mg/kg, about 7 mg/kg, about 8 mg/kg , about 9 mg/kg, about 10 mg/kg, about 20 mg/kg, about 30 mg/kg, about 40 mg/kg, about 50 mg/kg, about 60 mg/kg, about 70 mg/kg, about 80 mg/kg, about 90 mg/kg, about 100 mg/kg, about 110 mg/kg, about 120 mg/kg, about 130 mg/kg, about 140 mg/kg, or about 150 mg/kg.

In some embodiments, a composition that lowers neutralizing antibodies reduces and/or inhibits complement activation. For example, the composition can cleave neutralizing antibodies, preventing Clq from binding to antigen-antibody complexes (eg, AAV-antibody complexes). By reducing and/or inhibiting complement activation, downstream processes such as recruitment of inflammatory cells and opsonization (eg of AAV) in the complement cascade are prevented. In some embodiments, treating the subject with a composition that reduces neutralizing antibodies prior to treatment with AAV prevents complement activation in the patient upon administration of AAV, and in some embodiments, complement activation is at least 50%, at least 60% upon administration of AAV %, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%. In some embodiments, treatment of the patient with a composition that reduces neutralizing antibodies concurrently with treatment with AAV prevents complement activation in the patient due to treatment with AAV, and in some embodiments, complement activation is at least 50%, at least 60% , at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%. In some embodiments, treatment of the patient with one of the compositions that reduces neutralizing antibodies after AAV treatment reduces complement activation in the patient, and in some embodiments, complement activation is at least 50%, at least 60%, at least 70%, at least 80 %, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%.

In some embodiments, the compositions described herein comprise one or more pharmaceutically acceptable carriers, excipients and/or vehicles, e.g., solvents, buffers, solutions, dispersion media, coatings, antibacterial and antifungal agents, isotonicity. substances and absorption delaying agents. In some embodiments, pharmaceutically acceptable carriers, excipients and/or vehicles are saline, buffered saline, dextrose, water, glycerol, sterile isotonic aqueous buffer, phosphate buffered solution, amino acid-based buffer, bi carbonate buffered solutions and combinations thereof. In some embodiments, pharmaceutically acceptable carriers, excipients and/or vehicles are phosphate buffered saline, sterile saline, lactose, sucrose, calcium phosphate, dextran, agar, pectin, peanut oil, sesame oil, pharmaceutical grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, polyols (eg, glycerol, propylene glycol and liquid polyethylene glycol, etc.) or suitable mixtures thereof. In some embodiments, the compositions described herein further comprise a minor amount of an emulsifying or wetting agent or a pH buffering agent. Formulations of the compositions described herein may be prepared, for example, in the form of lyophilized powders, slurries, aqueous solutions or suspensions by mixing with physiologically acceptable carriers, excipients or stabilizers (e.g., Hardman, (2001) Goodman and Gilman's The Pharmacological Basis of Therapeutics, McGraw-Hill, New York, NY; Gennaro (2000) Remington: The Science and Practice of Pharmacy, Lippincott, Williams, and Wilkins, New York, NY; (eds.) (1993) Pharmaceutical Dosage Forms: Parenteral Medications, Marcel Dekker, NY; Lieberman, et al. (eds.) (1990) Pharmaceutical Dosage Forms: Tablets, Marcel Dekker, NY; Lieberman, et al (eds.) (1990) Pharmaceutical Dosage Forms: Disperse Systems, Marcel Dekker, NY; see Weiner and Kotkoskie (2000) Excipient Toxicity and Safety, Marcel Dekker, Inc., New York, NY).

In some embodiments, the compositions described herein contain conventional pharmaceutical ingredients such as preservatives, or chlorobutanol, potassium sorbate, sorbic acid, sulfur dioxide, propyl gallate, parabens, ethyl vanillin, glycerin, phenol, parachlorophenol or and a chemical stabilizer such as albumin. In some embodiments, the compositions described herein include antibacterial and antifungal agents such as parabens, chlorobutanol, phenol, sorbic acid or thimerosal; isotonic agents such as sugars or sodium chloride; and/or absorption delaying agents such as aluminum monostearate and gelatin.

In some embodiments, the compositions herein are formulated in a neutral or salt form. Pharmaceutically-acceptable salts include, for example, acids (formed with free amino groups of proteins) derived from, for example, inorganic acids, such as hydrochloric or phosphoric acid, or organic acids, such as acetic, oxalic, tartaric, mandelic, and the like. addition salts, and the like. In some embodiments, the salt formed with the free carboxy group of the protein is an inorganic base (e.g., sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide or ferric hydroxide) or an organic base (e.g., isopropylamine, trimethylamine) , histidine, procaine) and the like.

In some embodiments, the composition is in solid form, such as a lyophilized powder, suitable for reconstitution, liquid solution, suspension, emulsion, tablet, pill, capsule, sustained release formulation or powder form. In some embodiments, compositions can be formulated for delivery using liposomes, nanocapsules, microparticles, microspheres, liquid particles, vesicles, liposomes, nanospheres, nanoparticles, and the like.

Dosage and mode of administration of compositions for reducing neutralizing antibodies

Administration of any one of the compositions described herein for lowering neutralizing antibodies to a biologic can be effected by injection, infusion, or a combination thereof. A pharmaceutical composition comprising any of the compositions described herein may be from about 0.05 mg/kg to about 150 mg/kg, for example, from about 0.1 mg/kg, including all values and subranges intervening therebetween. , about 0.5 mg/kg, about 1 mg/kg, about 2 mg/kg, about 3 mg/kg, about 4 mg/kg, about 5 mg/kg, about 6 mg/kg, about 7 mg/kg, about 8 mg/kg, about 9 mg/kg, about 10 mg/kg, about 20 mg/kg, about 30 mg/kg, about 40 mg/kg, about 50 mg/kg, about 60 mg/kg, about 70 mg /kg, about 80 mg/kg, about 90 mg/kg, about 100 mg/kg, about 110 mg/kg, about 120 mg/kg, about 130 mg/kg, about 140 mg/kg or about 150 mg/kg It can be administered at a dosage of

A therapeutically effective amount of any one of the compositions described herein may be given in one dose, but is not limited to one dose. That is, the administration may be made 1 to 50 times, for example 2 times, 5 times, 10 times, 15 times, 20 times, 25 times, 30 times, 35 times, 40 times, 45 times or 50 times. When two or more doses are administered in the present invention, the dosing interval is from about 1 minute to about 1 month, for example, about 1 minute, about 2 minutes, about 3 minutes, including all values and subranges existing between the values. About 4 minutes, about 5 minutes, about 6 minutes, about 7 minutes, about 8 minutes, about 9 minutes, about 10 minutes, about 20 minutes, about 40 minutes, about 1 hour, about 2 hours, about 3 hours, about 4 time, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 15 hours, about 20 hours, about 24 hours, about 2 days, about 5 days, about 10 days, It may be about 15 days, about 20 days. The present invention is not limited to equally spaced dosing intervals, but non-uniformly spaced dosing, eg, administrations on days 1, 4, 7 and 25, to provide only non-limiting examples. It also includes a priming plan schedule consisting of

For example, once/week, 2 times/week, 3 times/week, 4 times/week, 5 times/week, 6 times/week, 7 times/week, 1 time/2 weeks, 1 time/3 weeks, Dosing schedules such as once/4 weeks, once/5 weeks, and the like are also available in the present invention. The dosing schedule may be from about 1 day to about 1 year, eg, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 2 months, 3 weeks, including all numbers and subranges that exist therebetween. months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months and 12 months.

Examples of cycles of the dosing schedule described above are provided. The cycle is approximately, for example, every 7 days; every 14 days; every 21 days; every 28 days; every 35 days; every 42 days; every 49 days; every 56 days; every 63 days; It can be repeated every 70 days, and so on. There may be non-administration intervals between cycles, which intervals are approximately, for example, 7 days; 14 days; 21 days; 28 days; 35 days; 42 days; 49 days; 56 days; 63 days; 70 days or the like.

The compositions described herein may be administered in conjunction with one or more additional therapeutic agents. Methods for co-administration with additional therapeutic agents are well known in the art (Hardman, et al. (eds.) (2001) Goodman and Gilman's The Pharmacological Basis of Therapeutics, 10th ed., McGraw-Hill, New York, NY; Poole and Peterson (eds.) (2001) Pharmacotherapeutics for Advanced Practice: A Practical Approach, Lippincott, Williams & Wilkins, Phila., Pa.; Chabner and Longo (eds.) (2001) Cancer Chemotherapy and Biotherapy , Lippincott, Williams & Wilkins, Phila., Pa.).

Subjects to be treated herein include mammals such as humans and non-human primates. In some embodiments, the subject may be selected from a human, a non-human primate, a cow, a sheep, a goat, a horse, a cat, a dog, and a Lemur.

neutralizing antibodies to biologics how to degrade

The present invention provides a method for promoting degradation of a recombinant biologic or drug substance, and (a) an antibody to the recombinant biologic or drug substance; and/or (b) neutralizing a recombinant biologic or drug substance in an individual comprising administering to the individual a therapeutically effective amount of any one composition described herein that reduces binding of said antibody to an Fc receptor. A method for lowering an antibody is provided.

In some embodiments, a method of reducing the content of neutralizing antibodies to a recombinant adeno-associated virus (AAV) vector in an individual comprises administering to the individual a therapeutically effective amount of a composition that promotes degradation of the neutralizing antibody.

In some embodiments, the method of preparing a subject for treatment with a recombinant adeno-associated virus (AAV) vector comprises (a) promoting degradation of neutralizing antibodies to the AAV vector, and/or (b) binding to the Fc receptor. and administering to the subject a therapeutically effective amount of a composition that reduces binding of a neutralizing antibody.

In some embodiments, the method of treating a subject in need with a recombinant adeno-associated virus (AAV) vector comprises: (i) (a) promoting degradation of neutralizing antibodies to the AAV vector, and/or (b) binding to the Fc receptor. administering to the subject a therapeutically effective amount of a composition for reducing binding of a neutralizing antibody to; and (ii) administering the AAV vector to the subject in a therapeutically effective amount.

In some embodiments, the method of treating an individual previously treated with a first recombinant AAV with a second recombinant adeno-associated virus (AAV) vector comprises: (i) (a) a first and/or second recombinant AAV vector. administering to the subject a therapeutically effective amount of a composition that promotes the degradation of the neutralizing antibody to, and/or (b) reduces the binding of the neutralizing antibody to the Fc receptor; and (ii) administering to the subject a therapeutically effective amount of a second recombinant AAV vector.

In some embodiments, a method of lowering a neutralizing antibody to an adeno-associated virus (AAV) vector comprising a heterologous nucleic acid in an individual comprises a therapeutically effective amount of an AAV vector, and (a) an antibody to the AAV vector or a heterologous nucleic acid. to promote degradation of the recombinant protein encoded by; and/or (b) administering to the subject a composition that reduces binding of the antibody to an Fc receptor.

In some embodiments, a method of lowering a neutralizing antibody to any one adeno-associated virus (AAV) vector described herein comprises a therapeutically effective amount of an AAV vector, and (a) an antibody or heterologous nucleic acid to the AAV vector. to promote degradation of the recombinant protein encoded by; and/or (b) administering to the subject any one composition described herein that reduces binding of the antibody to an Fc receptor.

A composition that (a) promotes degradation of a neutralizing antibody to the first and/or second recombinant AAV vector, and/or (b) reduces binding of the neutralizing antibody to an Fc receptor, comprises an antibody-degrading enzyme or fragment thereof. may include In some embodiments, the composition comprises a vector comprising a polynucleotide encoding an antibody-degrading enzyme or fragment thereof. In some embodiments, the aforementioned antibody-degrading enzymes or fragments thereof may have cysteine protease activity. In some embodiments, the antibody-degrading enzymes specifically cleave IgG. In some embodiments, the antibody-degrading enzyme or fragment thereof is from the genus Streptococcus. In some embodiments, the antibody-degrading enzyme comprises an amino acid sequence that is at least 90% or at least 95% identical to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the antibody-degrading enzyme comprises the amino acid sequence of SEQ ID NO:1.

In some embodiments, the composition may comprise a fusion protein comprising a first protein and a second protein, wherein the first protein is an antibody-degrading enzyme or fragment thereof. In some embodiments, the first protein and the second protein are separated by a linker. In some embodiments, the second protein is an IgG protease.

The composition may be administered via a systemic route (eg, intravenous, intraarticular or intralymphatic). In some embodiments, the composition is delivered topically (eg, intramuscularly, intradermally, subcutaneously, topically). In some embodiments, the composition is administered directly to a site known to contain neutralizing antibodies, such as cerebrospinal fluid (CSF). The composition may also include a pharmaceutically acceptable carrier and/or diluent.

In some embodiments, the subject is administered about 0.1 mg/kg to about 100 mg/kg of the antibody-degrading enzyme or fragment thereof. In some embodiments, the subject is administered about 0.1 mg/kg to about 100 mg/kg of the fusion protein. In some embodiments, the subject is administered about 0.05 mg/kg to about 150 mg/kg of an antibody-degrading enzyme or fragment thereof, or fusion protein, including, for example, all numbers and subranges that exist between the numbers; About 0.1 mg/kg, about 0.5 mg/kg, about 1 mg/kg, about 2 mg/kg, about 3 mg/kg, about 4 mg/kg, about 5 mg/kg, about 6 mg/kg, about 7 mg/kg, about 8 mg/kg, about 9 mg/kg, about 10 mg/kg, about 20 mg/kg, about 30 mg/kg, about 40 mg/kg, about 50 mg/kg, about 60 mg/kg kg, about 70 mg/kg, about 80 mg/kg, about 90 mg/kg, about 100 mg/kg, about 110 mg/kg, about 120 mg/kg, about 130 mg/kg, about 140 mg/kg, or about 150 mg/kg.

In some embodiments, the neutralizing antibody to be degraded and/or degraded comprises IgG, IgM, IgE and/or IgA. In some embodiments, the neutralizing antibody comprises IgG. In some embodiments, the antibody is a neutralizing antibody against an AAV vector comprising a transgene. In some embodiments, the antibody binds to a recombinant protein encoded by a transgene. In some embodiments, the antibody binds to an adeno-associated viral capsid protein or a virion thereof.

In some embodiments, the recombinant AAV vector is an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh8, AAVrh10, AAVrh32.33, AAVrh74, avian AAV or bovine AAV vector. am. In some embodiments, the recombinant AAV vector has a sequence of any one of SEQ ID NOs: 2-12, or at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical thereto. capsid proteins. In some embodiments, the AAV vector is a wild-type AAV vector. In some embodiments, the AAV vector is a mutant AAV vector. In some embodiments, the AAV vector is a wild-type AAV1 vector. In some embodiments, the AAV vector is a wild-type AAV2 vector. In some embodiments, the AAV vector is a wild-type AAV4 vector. In some embodiments, the AAV vector is a wild-type AAV8 vector. In some embodiments, the AAV vector is a wild-type AAV9 vector. In some embodiments, the AAV vector is a mutant AAV1 vector. In some embodiments, the AAV vector is a mutant AAV2 vector. In some embodiments, the AAV vector is a mutant AAV4 vector. In some embodiments, the AAV vector is a mutant AAV8 vector. In some embodiments, the AAV vector is a mutant AAV9 vector. In some embodiments, the recombinant AAV vector comprises a heterologous nucleic acid encoding a therapeutic protein or therapeutic RNA.

In some embodiments, the methods described herein comprise lowering the interaction between the antibody and its cognate receptor on the cell surface. Such methods can expand a cohort of patients suitable for gene therapy, and also allow AAV re-dosing/re-dosing in patients previously treated with AAV vectors.

In some embodiments of the methods herein, the subject is administered an AAV vector with the composition. In some embodiments, the AAV vector is administered after administration of the composition to the subject. In some embodiments, the AAV vector is administered prior to administering the composition to the subject. In some embodiments, the method further comprises administering the second AAV vector comprising the second heterologous nucleic acid one or more times as an additional dose or as a second dose. In some embodiments, the first AAV vector and the second AAV vector comprise the same AAV capsid protein. In some embodiments, the first AAV vector and the second AAV vector comprise different AAV capsid proteins.

In some embodiments, the method promotes degradation of an antibody to an AAV vector. In some embodiments, the level of the antibody ranges from about 95% to about 0.01% of the antibody level in a control subject (e.g., about 90%, about 85%, including all values and subranges therebetween, About 80%, about 75%, about 70%, about 65%, about 60%, about 55%, about 50%, about 45%, about 40%, about 35%, about 30%, about 25%, about 20 %, about 15%, about 10%, about 5%, about 1%, about 0.1%, or about 0.01%). As used herein, a control individual is an individual administered with a recombinant biologic, such as an AAV vector, but not administered with any one of the compositions described herein. In some embodiments, in such methods, after administration of the composition, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 99% is degraded.

In some embodiments, the subject has been previously administered the recombinant protein. Accordingly, in some embodiments, administration of any one of the compositions described herein reduces circulating levels of antibodies produced prior to administration of the recombinant protein in an individual.

Importantly, the compositions and method(s) according to the present invention may be used in conjunction with other pharmacological or interventional approaches capable of reducing antibodies.

Recombinant Virus Vectors

In some embodiments, the vectors described herein are usable for delivery of heterologous nucleic acids to cells in vitro, ex vivo and in vivo. In some embodiments, the vector is a viral vector, eg, an AAV vector. In particular, it is advantageously possible to employ viral vectors for delivery of nucleic acids to animal cells, for example mammalian cells. In some embodiments, a viral vector comprises a recombinant viral capsid surrounding a heterologous nucleic acid, eg, an AAV capsid. In some embodiments, the recombinant viral capsid comprises a recombinant capsid protein, eg, a recombinant AAV capsid protein. Detailed descriptions of viral vectors, viral capsids and/or capsid proteins usable in the present invention are provided in International Applications PCT/US2019/025617, PCT/US2019/025584 and PCT/US2019/025610, each of which is set forth in its entirety. Incorporated herein by reference for all purposes.

In some embodiments, the AAV vector is AAV1, AAV2, AAV3, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh.8, AAVrh.10, AAVrh.32.33, AAVrh74, bovine capsid proteins of AAV serotypes selected from AAV, avian AAV or any other AAV currently known or to be identified in the future. In some embodiments, the AAV capsid protein is chimeric.

In some embodiments, the capsid protein is an AAV capsid protein (VP1, VP2 and/or VP3) comprising a modification (eg, a substitution) in the amino acid sequence, and the viral capsid and viral vector comprise a modified AAV capsid protein. In some embodiments, the modifications described herein can confer one or more desirable properties to a viral vector comprising a modified AAV capsid protein, including, but not limited to, the ability to evade neutralizing antibodies.

In some embodiments, the AAV capsid protein comprises one or more amino acid substitutions, wherein the one or more substitutions modify one or more antigenic sites of the AAV capsid protein. By modifying the one or more antigenic sites, inhibition of binding of the antibody to the one or more antigenic sites and/or neutralizing inhibition of infectivity of the viral particle comprising the AAV capsid protein is achieved. In some embodiments, the modification of one or more antigenic sites inhibits antibody binding to one or more antigenic sites. In some embodiments, the modified antigenic site may prevent the antibody from binding to, or recognizing, or neutralizing the AAV capsid, wherein the antibody is an IgG (IgG1, IgG2a, IgG2b, IgG3, etc.), IgM, IgE or IgA. In some embodiments, modification of one or more antigenic sites neutralizes the infectivity of a viral particle comprising an AAV capsid protein.

The one or more amino acid substitutions may be made in one or more antigenic footprints identified by cold-electron microscopy experiments and/or peptide epitope mapping of an AAV-antibody complex comprising an AAV capsid protein. In some embodiments, the one or more antigenic sites are a common antigenic motif or CAM as described in WO 2017/058892, which is incorporated herein by reference in its entirety. In some embodiments, the antigenic site is of an AAV capsid protein such as VR-I, VR-II, VR-III, VR-IV, VR-V, VR-VI, VR-VII, VR-VIII, VR-IX. It is present in the variable part (VR). In some embodiments, the one or more antigenic sites are in the HI loop of the AAV capsid protein.

In some embodiments, the amino acid substitution replaces any 6, 7 or 8 amino acids in an AAV capsid protein derived from any of the following serotypes: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6 , AAV7, AAV8, AAV9, AAVrh8, AAVrh10, AAV10, AAV11, AAV12, AAVrh32.22, bovine AAV or avian AAV. In some embodiments, the substitution introduces a deletion in the AAV capsid sequence. For example, a sequence of 6, 7, 8 or 9 amino acids replaces 7, 8, 9 or 10 amino acids respectively of the original amino acid capsid sequence. In some embodiments, the substitution introduces an insertion into the AAV capsid sequence. For example, a sequence of 6, 7, 8 or 9 amino acids replaces 5, 6, 7 or 8 amino acids respectively of the original amino acid capsid sequence.

In some embodiments, the one or more substitutions of one or more antigenic sites will introduce one or more antigenic sites derived from a capsid protein of a first AAV serotype into a capsid protein of a second AAV serotype that is different from the first AAV serotype. can

As used herein, “substitution” may refer to a substitution of a single amino acid, or a substitution of two or more amino acids. For example, in some embodiments, the capsid proteins of the present disclosure contain at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, etc. of a single amino acid. It may include substitutions. In some embodiments, a capsid protein of the invention comprises one or more substitutions of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 contiguous amino acids; Likewise, it may include one or more substitutions of a plurality of contiguous amino acids.

Furthermore, in the embodiments described herein in which an amino acid residue is substituted with any amino acid residue other than an amino acid residue present in the wild-type or native amino acid sequence, any other amino acid residue is selected from any naturally occurring amino acid residue known in the art. or non-natural amino acid residues (see, eg, Tables 3 and 4 ). In some embodiments, a substitution can be a conservative substitution, and in some embodiments, a substitution can be a non-conservative substitution.

In some embodiments, the AAV capsid protein comprises a first amino acid substitution and a second amino acid substitution, wherein the first amino acid substitution and the second amino acid substitution each modify different antigenic sites on the AAV capsid protein. In some embodiments, the AAV capsid protein comprises a first amino acid substitution, a second amino acid substitution, and a third amino acid substitution, wherein the first amino acid substitution, the second amino acid substitution, and the third amino acid substitution are each different from each other on the AAV capsid protein. modify antigenic sites.

Any of the AAV capsids described herein may further comprise a modification (eg, substitution or deletion) in the HI loop. The HI loop is the major domain of the AAV capsid surface that extends from each viral protein (VP) subunit and overlaps the neighboring quintuple VP, between the β strands βH and βI. In some embodiments, the AAV capsid comprises 1, 2, 3, 4, 5, 6, 7 or 8 amino acid substitutions in the HI loop. In some embodiments, the AAV capsid protein comprises 1, 2, 3 or 4 amino acid substitutions, wherein each substitution modifies a different antigenic site on the AAV capsid protein, wherein at least one of the amino acid substitutions is Modify the HI loop of the capsid protein. In some embodiments, the AAV capsid protein comprises first, second, third and fourth amino acid substitutions.

In some embodiments, the AAV capsid proteins described herein are encoded by, and expressed from, a nucleotide sequence or an expression vector comprising the same. The nucleotide sequence may be a DNA sequence or an RNA sequence.

In some embodiments, the modified capsid protein is prepared by modifying the capsid protein of any AAV currently known or to be developed in the future. In addition, the AAV capsid protein to be modified is a naturally occurring AAV capsid protein (eg, AAV2, AAV3a or 3b, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10 or AAV11 capsid protein or any AAV shown in Table 2) ), but is not limited thereto. Those of ordinary skill in the art will appreciate that various manipulations of AAV capsid proteins are known in the art and that the present invention is not limited to modifications of naturally occurring AAV capsid proteins. For example, the capsid protein to be modified may be a naturally occurring AAV (e.g., from a naturally occurring AAV capsid protein, e.g., AAV2, AAV3a, AAV3b, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11 , AAV12 or any other AAV currently known or to be discovered in the future). In some embodiments, the capsid protein may be a chimeric capsid protein. In some embodiments, the capsid protein may be an engineered AAV, eg, AAV2i8, AAV2g9, AAV-LK03, AAV7m8, AAV Anc80, AAV PHP.B. Such AAV capsid proteins are also included within the scope of the present invention.

Thus, in some embodiments, the AAV capsid protein to be modified may be derived from a naturally occurring AAV, but inserted and/or substituted in the capsid protein and/or modified by deletion of one or more amino acids (e.g., and may further comprise one or more foreign sequences (exogenous to the native virus). In some embodiments, modifications to the AAV capsid protein are "selective" modifications. This approach contrasts with previous methods of exchanging subunit whole or large domains between AAV serotypes (see, eg, International Patent Publication No. WO 00/28004 and Hauck et al., (2003) J. See Virology 77:2768-2774). In certain embodiments, "optional" modifications are no more than about 20, no more than about 18, no more than about 15, no more than about 12, no more than about 10, no more than about 9, no more than about 8, no more than about 7 contiguous amino acids. no more than about 6, no more than about 5, no more than about 4, or no more than about 3 insertions and/or substitutions and/or deletions. The modified capsid proteins and capsids of the present invention may further comprise any other modifications currently known or to be identified in the future.

Thus, specific AAV capsid proteins herein (eg, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10 or AAV11 capsid proteins or capsid proteins, etc. derived from any AAV mentioned in Table 2) Where reference is made, it is intended to encompass not only the native capsid protein, but also the capsid protein having variations other than the modifications herein. Such mutations include substitutions, insertions and/or deletions. In certain embodiments, the capsid protein contains 1, 2, 3, 4, 5, 6 internally inserted amino acids (other than insertions referred to herein) compared to the native AAV capsid protein sequence. , 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20, less than 20, less than 30 , less than 40, less than 50, less than 60, or less than 70. In embodiments, the capsid protein has 1, 2, 3, 4, 5, 6 amino acid substitutions (other than the amino acid substitutions recited herein) compared to the native AAV capsid protein sequence. , 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20, less than 20, less than 30 , less than 40, less than 50, less than 60, or less than 70, and in embodiments of the invention, the capsid protein, compared to the native AAV capsid protein sequence other) amino acid deletions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 , 16, 17, 18, 19 or 20, less than 20, less than 30, less than 40, less than 50, less than 60 or less than 70.

Methods for determining sequence similarity or identity between two or more amino acid sequences are known in the art. Sequence similarity or identity can be determined by, but not limited to, Smith & Waterman, Adv. Appl. Math. 2, 482 (1981), the local sequence identity algorithm, Needleman & Wunsch, J Mol. Biol. 48,443 (1970), the sequence identity alignment algorithm, Pearson & Lipman, Proc. Natl. Acad. Sci. USA 85, 2444 (1988), computational processing of these algorithms (GAP, BESTFIT, FASTA and TFASTA, Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, WI), Devereux et al., Nucl . Acid Res. 12, 387-395 (1984), using standard techniques known in the art, such as by the best fit sequence program or assay.

Other suitable algorithms are described in Altschul et al., J Mol. Biol. 215, 403-410, (1990) and Karlin et al., Proc. Natl. Acad. Sci. USA 90, 5873-5787 (1993). Particularly useful BLAST programs are Altschul et al., Methods in Enzymology, 266, 460-480 (1996); This is the WU-BLAST-2 program obtained from http://blast.wustl/edu/blast/README.html. WU-BLAST-2 uses several search parameters, optionally set to default values. The parameters are dynamic values and are set by their own programs according to the composition of the specific sequence and the configuration of the specific database to search for the target sequence; These values can be adjusted to increase sensitivity.

In addition, additionally available algorithms are described in Altschul et al, (1997) Nucleic Acids Res. 25, 3389-3402, is a gapped BLAST.

In some embodiments, the AAV vector comprises an AAV capsid and an AAV genome and has a phenotype that avoids neutralizing antibodies. In addition, the vectors or AAV viral particles described herein may have enhanced or maintained transduction efficiency, with a phenotype that avoids neutralizing antibodies.

The viral vector according to the present invention can be produced by any method known in the art, for example by expression using a baculovirus system.

In some embodiments of the invention, the viral capsid comprises a targeting sequence (eg, substituted or inserted in the viral capsid) that directs the viral capsid to interact with cell-surface molecules present on the desired target tissue(s). which may be a targeted viral capsid. For example, a viral capsid of the present invention has a relative tropism towards a particular target tissue of interest (eg, liver, skeletal muscle, heart, diaphragm muscle, kidney, brain, stomach, intestine, skin, endothelial cells and/or lung). may not be valid. Targeting sequences can advantageously be incorporated into such low-efficiency-transduction vectors to confer the desired tropism to the viral capsid and optionally to specific tissue(s). Vectors comprising AAV capsid proteins, capsids and targeting sequences are described, for example, in International Patent Publication No. WO 00/28004. For another example, see Wang et al., Annu Rev Biophys Biomol Struct. 35:225-49 (2006)), wherein one or more non-naturally occurring amino acids are at an orthogonal site as a way to re-apply a desired low-efficiency-transduction vector to the target tissue(s). may be incorporated into the AAV capsid subunits herein. These non-natural amino acids may advantageously include, but are not limited to, glycans (mannose - targeting dendritic cells); neuropeptides for targeted delivery to RGD, bombesin or specific cancer cell types; To chemically link molecules of interest to AAV capsid proteins, including RNA aptamers or peptides selected from phage display that target specific cell surface receptors such as growth factor receptors, integrins, and the like.

In some embodiments of the invention, the capsid protein, viral capsid or vector of the invention has an equivalent or enhanced transduction efficiency with respect to the transduction efficiency of the AAV serotype from which the capsid protein, viral capsid or vector of the invention is derived. can have In some embodiments of the invention, the capsid protein, viral capsid or vector of the invention may have reduced transduction efficiency relative to the transduction efficiency of the AAV serotype from which the capsid protein, viral capsid or vector of the invention is derived. have. In some embodiments of the present invention, the capsid protein, viral capsid or vector of the present invention may have an equivalent or enhanced tropism with respect to the tropism of the AAV serotype from which the capsid protein, viral capsid or vector of the present invention is derived. . In some embodiments of the invention, a capsid protein, viral capsid or vector of the invention may have a modified or different tropism with respect to the tropism of the AAV serotype from which the capsid protein, viral capsid or vector of the invention is derived. In some embodiments of the invention, capsid proteins, viral capsids or vectors of the invention have or can be engineered to have tropism for brain tissue. In some embodiments of the invention, the capsid proteins, viral capsids or vectors of the invention have or can be engineered to have tropism for liver tissue.

One of ordinary skill in the art will recognize that for some AAV capsid proteins, the modifications will be insertions and/or substitutions, depending on whether some or all of the amino acid positions in question are present in the virus or, alternatively, all absent. As described elsewhere herein, those of ordinary skill in the art will readily be able to ascertain the amino acid position(s) of interest using well-known techniques.

AAV virus vector

In some embodiments, a viral vector comprises a modified AAV capsid comprising a modified capsid subunit of the invention and a vector genome. For example, in some embodiments, the viral vector comprises (a) a modified viral capsid (eg, a modified AAV capsid) comprising a modified capsid protein of the invention; and (b) a heterologous nucleic acid comprising a terminal repeat sequence (eg, AAV TR), wherein the heterologous nucleic acid comprising a terminal repeat sequence is surrounded by a modified viral capsid. The nucleic acid may optionally comprise two terminal repeats (eg, two AAV TRs).

In some embodiments, the viral vector of the invention (i) has reduced transduction in the liver compared to the level of transduction by the viral vector in which the capsid protein is not modified; (ii) systemic transduction of the capsid protein by the viral vector in an animal subject is enhanced as compared to levels observed with the unmodified viral vector; (iii) the capsid protein has enhanced migration across endothelial cells compared to the level of migration by the unmodified viral vector; and/or (iv) transduction into muscle tissue (eg, skeletal muscle, myocardium and/or diaphragm muscle) is selectively enhanced, (v) transduction into liver tissue is selectively enhanced, and/or (vi) reduced transduction into brain tissue (eg, neurons) compared to the level of transduction with the viral vector in which the capsid protein is not modified. In certain embodiments, the viral vector is systemically transduced into the liver.

In some embodiments, the viral vector is a recombinant viral vector comprising a heterologous nucleic acid or functional RNA encoding a polypeptide of interest. In some embodiments, the nucleic acid is a polypeptide encoding nucleic acid, including a therapeutic (eg, for medical or veterinary use) or immunogenic (eg, for a vaccine) polypeptide or RNA.

Alternatively, the immunogenic polypeptide may be any tumor or cancer cell antigen. Optionally, the tumor or cancer antigen is expressed on the surface of the cancer cell.

Those of ordinary skill in the art will appreciate that heterologous nucleic acids can be operably combined with appropriate regulatory sequences. For example, a heterologous nucleic acid can be operably combined with expression control factors such as transcriptional/translational control signals, origins of replication, polyadenylation signals, internal lysosomal entry points (IRES), promoters and/or enhancers, and the like. In addition, controlled expression of the heterologous nucleic acid(s) of interest can be achieved at the post-translational level, for example, in the presence or absence of oligonucleotides, small molecules and/or other compounds that selectively block splicing activity at specific sites. By controlling the selective splicing of various introns by

Those of ordinary skill in the art will appreciate that a variety of promoter/enhancer factors are available depending on the desired tissue-specific expression and level. Promoters/enhancers can be constitutive or inducible depending on the desired expression pattern. Promoters/enhancers may be native or foreign, and may be native or synthetic sequences. By foreign, it is meant that the transcription initiation is not found in the wild-type host into which it is introduced.

In certain embodiments, the promoter/enhancer factor may be native to the target cell or subject to be treated. In an exemplary embodiment, the promoter/enhancer factor may be native to a heterologous nucleic acid sequence. Promoter/enhancer factors are generally defined to function in the target cell(s) of interest. Furthermore, in certain embodiments, the promoter/enhancer factor is a mammalian promoter/enhancer factor. Promoter/enhancer factors may be constitutive or inducible.

Inducible expression regulators are typically beneficial in applications where it is desirable to provide control of overexpression of the heterologous nucleic acid sequence(s). Inducible promoter/enhancer factors for gene transfer may be tissue-specific or tissue-preferred promoter/enhancer factors, and may be muscle-specific or preferential (such as heart, skeletal muscle and/or smooth muscle specificity or preference), nervous tissue specific or preferred (such as brain-specific or preferred), eye-specific or preferred (such as retina-specific and corneal-specific), liver-specific or preferred, bone marrow-specific or preferred, pancreatic-specific or preferred specific, spleen-specific or preferred, and lung-specific or preferred promoter/enhancer factors. Other inducible promoter/enhancer factors include hormone-inducible factors and metal-inducible factors. Examples of inducible promoter/enhancer factors include, but are not limited to, Tet on/off factor, RU486-inducible promoter, ecdyson-inducible promoter, rapamacin-inducible promoter, and metallothionein promoter. do.

Viral vectors according to the present invention provide a means for delivery of heterologous nucleic acids to a wide range of cells, including dividing and non-dividing cells. Viral vectors can be employed to deliver a nucleic acid of interest to cells in vitro, for example to produce polypeptides in vitro or for ex vivo gene therapy. Viral vectors are additionally available in methods of delivering nucleic acids to a subject in need thereof, for example, to express immunogenic or therapeutic polypeptides or functional RNAs. In this way, polypeptides or functional RNAs can be produced in vivo in a subject. Since the subject is deficient for the polypeptide, the subject may be in need of the polypeptide. In addition, the present methods may be practiced, as the production of polypeptides or functional RNAs in an individual may confer some beneficial effect.

The viral vectors of the present invention can be used to deliver heterologous nucleic acids or functional RNAs encoding polypeptides to treat and/or prevent any disease condition in which delivery of a therapeutic polypeptide or functional RNA would be beneficial. Gene delivery has practical use in identifying and providing therapies for disease states. Defective genes are known and there are some cloned genetic disorders. In general, the diseases described above fall into two classes: those that are usually inherited in a recessive manner, usually enzyme deficient, and those that may involve regulatory or structural proteins and are typically inherited in a dominant manner. For deficient diseases, gene transfer can be used to deliver normal genes to affected tissues as an alternative therapy, as well as antisense mutations to build animal disease models. In the case of a disproportionate disease state, gene transfer can be used to build the disease state in a model system, which can then be used in an attempt to counter the disease state. Thus, the viral vector according to the present invention makes it possible to treat and/or prevent genetic diseases.

The viral vector according to the present invention can also be utilized to supply functional RNA to cells in vitro or in vivo. The functional RNA can be, for example, a non-coding RNA. In some embodiments, expression of a functional RNA in a cell may attenuate expression of a particular target protein by the cell. Accordingly, the functional RNA may be administered to reduce the expression of a specific protein in an individual in need. In some embodiments, expression of a functional RNA in a cell may enhance expression of a particular target protein by the cell. Therefore, by administering functional RNA, it is possible to increase the expression of a specific protein in a subject in need. In some embodiments, expression of a functional RNA can control splicing of a particular target RNA in a cell. Accordingly, by administering a functional RNA, splicing of a specific RNA can be controlled in a subject in need. In some embodiments, expression of a functional RNA in a cell can control the function of a particular target protein by the cell. That is, by administering a functional RNA, it is possible to control the function of a specific protein in a subject in need. In addition, functional RNA can be administered to cells in vitro to modulate gene expression and/or cell physiological properties, for example to optimize cell or tissue culture systems or in screening methods.

Alternatively, the viral vector can be administered to the cells ex vivo, and the mutated cells are administered to the subject. A viral vector comprising a heterologous nucleic acid is introduced into a cell, and the cell is administered to a subject, wherein the heterologous nucleic acid encoding the immunogen is expressed to induce an immune response to the immunogen in the subject. In certain embodiments, the cell is an antigen-presenting cell (eg, a dendritic cell).

kit

Another aspect of the invention provides a kit for lowering and/or scavenging neutralizing antibodies and/or immunoglobulins to a recombinant biologic and/or drug substance in a subject, the kit comprising any one of the compositions described herein; It comprises, consists of, or consists essentially of the means of administration and instructions for use of the composition. In some embodiments, the reagent comprises an immunoglobulin G (IgG)-degrading enzyme.

listed by number implementations

Embodiments numbered below are included within the scope of the present invention.

1. A neutralizing antibody to a recombinant adeno-associated virus (AAV) vector in a subject in need thereof comprising administering to the subject a therapeutically effective amount of a composition that promotes degradation of the neutralizing antibody to the recombinant adeno-associated virus (AAV) vector How to lower the content of.

2. The method according to embodiment 1, wherein the neutralizing antibody is IgG, IgM, IgE or IgA.

3. The method of embodiment 2, wherein the neutralizing antibody is IgG.

4. Embodiments wherein the recombinant AAV vector is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh8, AAVrh10, AAVrh32.33, AAVrh74, avian AAV or bovine AAV vector A method according to any one of Examples 1-3.

5. The method according to embodiment 4, wherein the AAV vector is a wild-type AAV vector.

6. The method according to embodiment 4, wherein the AAV vector is a mutant AAV vector.

7. The method according to any one of embodiments 1-6, wherein the recombinant AAV vector comprises a heterologous nucleic acid encoding a therapeutic protein or therapeutic RNA.

8. at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 99% of the antibody in the subject administering the composition The method according to any one of embodiments 1-7, which is then degraded.

9. The method according to any one of embodiments 1-8, wherein the composition comprises an antibody-degrading enzyme or fragment thereof.

10. A method according to any one of embodiments 1-8, wherein the composition comprises a vector comprising a polynucleotide encoding an antibody-degrading enzyme or fragment thereof.

11. The method according to embodiment 9 or 10, wherein said antibody-degrading enzyme or fragment thereof has cysteine protease activity.

12. The method according to any one of embodiments 9-11, wherein the antibody-degrading enzyme specifically cleaves IgG.

13. The method according to any one of embodiments 9-12, wherein the antibody-degrading enzyme or fragment thereof is derived from the genus Streptococcus.

14. The method according to any one of embodiments 9-13, wherein the antibody-degrading enzyme comprises an amino acid sequence that is at least 90% or at least 95% identical to the amino acid sequence of SEQ ID NO: 1.

15. The method according to embodiment 14, wherein the antibody-degrading enzyme comprises the amino acid sequence of SEQ ID NO: 1.

16. The method according to any one of embodiments 1-15, wherein the composition comprises a fusion protein comprising a first protein and a second protein, wherein the first protein is an antibody-degrading enzyme or fragment thereof.

17. The method according to embodiment 16, wherein the first protein and the second protein are separated by a linker.

18. The method according to embodiment 16 or 17, wherein the second protein is an IgG protease.

19. The method according to any one of embodiments 9-15, wherein about 0.1 mg/kg to about 100 mg/kg of the antibody-degrading enzyme or fragment thereof is administered to the subject.

20. A method according to any one of embodiments 1-19, wherein administration reduces binding of said antibody to Fc receptors.

21. The method according to any one of embodiments 1-20, wherein the composition is administered intravenously.

22. The method according to any one of embodiments 1-21, wherein the composition comprises a pharmaceutically acceptable carrier and/or diluent.

23. The method according to any one of embodiments 1-22, wherein the subject is a human.

24. The method according to any one of embodiments 1-23, wherein the subject is treated with a recombinant adeno-associated virus (AAV) vector prior to administration of the composition.

25. The method according to any one of embodiments 1-23, wherein the subject is not treated with recombinant AAV prior to administration of the composition.

26. A recombinant adeno-adjunct comprising administering to the subject an effective amount of a composition that (a) promotes degradation of a neutralizing antibody to an AAV vector, and/or (b) reduces binding of the neutralizing antibody to an Fc receptor. A method of preparing a subject for treatment with a viral (AAV) vector.

27. The method according to embodiment 26, wherein the neutralizing antibody is IgG, IgM, IgE or IgA.

28. The method according to embodiment 27, wherein the neutralizing antibody is IgG.

29. Embodiments wherein the recombinant AAV vector is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh8, AAVrh10, AAVrh32.33, AAVrh74, avian AAV or bovine AAV vector A method according to any one of Examples 26-28.

30. The method according to embodiment 29, wherein the AAV vector is a wild-type AAV vector.

31. The method according to embodiment 29, wherein the AAV vector is a mutant AAV vector.

32. The method according to any one of embodiments 26-31, wherein the recombinant AAV comprises a heterologous nucleic acid encoding a therapeutic protein or therapeutic RNA.

33. At least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 99% of the antibody is present in the subject after administration of the composition. The method according to any one of embodiments 26-32, wherein

34. The method according to any one of embodiments 26-33, wherein the composition comprises an antibody-degrading enzyme or fragment thereof.

35. A method according to any one of embodiments 26-33, wherein the composition comprises a vector comprising a polynucleotide encoding an antibody-degrading enzyme or fragment thereof.

36. A method according to embodiment 34 or 35, wherein the antibody-degrading enzyme or fragment thereof has cysteine protease activity.

37. The method according to any one of embodiments 26-36, wherein the antibody-degrading enzyme specifically cleaves IgG.

38. The method according to any one of embodiments 26-37, wherein the antibody-degrading enzyme or fragment thereof is derived from the genus Streptococcus.

39. The method according to any one of embodiments 26-38, wherein the antibody-degrading enzyme comprises an amino acid sequence that is at least 90% or at least 95% identical to the amino acid sequence of SEQ ID NO: 1.

40. The method according to embodiment 30, wherein the antibody-degrading enzyme comprises the amino acid sequence of SEQ ID NO: 1.

41. A method according to any one of embodiments 26-40, wherein the composition comprises a fusion protein comprising a first protein and a second protein, wherein the first protein is an antibody-degrading enzyme or fragment thereof.

42. The method according to embodiment 41, wherein the first protein and the second protein are separated by a linker.

43. The method according to embodiment 41 or 42, wherein the second protein is an IgG protease.

44. The method according to any one of embodiments 34-43, wherein the subject is administered from about 0.1 mg/kg to about 100 mg/kg of the antibody-degrading enzyme or fragment thereof.

45. A method according to any one of embodiments 28-44, wherein the administering reduces binding of the antibody to the Fc receptor.

46. The method according to any one of embodiments 26-45, wherein the composition is administered intravenously.

47. The method according to any one of embodiments 26-46, wherein the composition comprises a pharmaceutically acceptable carrier and/or diluent.

48. The method according to any one of embodiments 26-47, wherein the subject is a human.

49. A method of treating a subject in need thereof with a recombinant adeno-associated virus (AAV) vector comprising the steps of:

(i) administering to the subject an effective amount of a composition that (a) promotes degradation of the neutralizing antibody to an AAV vector, and/or (b) reduces binding of the neutralizing antibody to an Fc receptor; and

(ii) administering to said subject an effective amount of an AAV vector.

50. The method according to embodiment 49, wherein the AAV vector is administered simultaneously with said composition.

51. The method according to embodiment 49, wherein the AAV vector is administered after administration of said composition.

52. The method according to embodiment 49, wherein the AAV vector is administered prior to administering said composition.

53. The method according to any one of embodiments 49-52, wherein the method further comprises administering to the subject a second AAV vector.

54. The method according to embodiment 53, wherein the AAV vector and the second AAV vector comprise an AAV capsid protein of the same serotype.

55. The method according to embodiment 53, wherein the AAV vector and the second AAV vector comprise AAV capsid proteins of different serotypes.

56. The method according to any one of embodiments 45-55, wherein the neutralizing antibody is IgG, IgM, IgE or IgA.

57. The method according to embodiment 56, wherein the neutralizing antibody is IgG.

58. The embodiment wherein the recombinant AAV vector is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh8, AAVrh10, AAVrh32.33, AAVrh74, avian AAV or bovine AAV vector. A method according to any one of Examples 49-57.

59. The method according to embodiment 58, wherein the AAV vector is a wild-type AAV vector.

60. The method according to embodiment 58, wherein the AAV vector is a mutant AAV vector.

61. The method according to any one of embodiments 49-60, wherein the recombinant AAV vector comprises a heterologous nucleic acid encoding a therapeutic protein or therapeutic RNA.

62. At least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 99% of the antibody is present in the subject after administration of the composition. The method according to any one of embodiments 49-61, wherein

63. The method according to any one of embodiments 49-62, wherein the composition comprises an antibody-degrading enzyme or fragment thereof.

64. The method according to any one of embodiments 49-62, wherein the composition comprises a vector comprising a polynucleotide encoding an antibody-degrading enzyme or fragment thereof.

65. The method according to embodiment 63 or 64, wherein the antibody-degrading enzyme or fragment thereof has cysteine protease activity.

66. The method according to any one of embodiments 63-65, wherein the antibody-degrading enzyme specifically cleaves IgG.

67. The method according to any one of embodiments 63-66, wherein the antibody-degrading enzyme or fragment thereof is derived from the genus Streptococcus.

68. The method according to any one of embodiments 63-67, wherein the antibody-degrading enzyme comprises an amino acid sequence that is at least 90% or at least 95% identical to the amino acid sequence of SEQ ID NO: 1.

69. The method according to embodiment 68, wherein the antibody-degrading enzyme comprises the amino acid sequence of SEQ ID NO: 1.

70. A method according to any one of embodiments 49-69, wherein the composition comprises a fusion protein comprising a first protein and a second protein, wherein the first protein is an antibody-degrading enzyme or fragment thereof.

71. The method according to embodiment 70, wherein the first protein and the second protein are separated by a linker.

72. A method according to embodiment 70 or 71, wherein the second protein is an IgG protease.

73. The method according to any one of embodiments 63-72, wherein the subject is administered from about 0.1 mg/kg to about 100 mg/kg of the antibody-degrading enzyme or fragment thereof.

74. A method according to any one of embodiments 49-73, wherein the administering reduces binding of the antibody to the Fc receptor.

75. The method according to any one of embodiments 49-74, wherein the composition is administered intravenously.

76. The method according to any one of embodiments 49-75, wherein the composition comprises a pharmaceutically acceptable carrier and/or diluent.

77. The method according to any one of embodiments 49-76, wherein the subject is a human.

78. A method of treating a second recombinant AAV in an individual previously treated with a first recombinant adeno-associated virus (AAV) vector comprising the steps of:

(i) administering to the subject an effective amount of a composition that (a) promotes degradation of the neutralizing antibody to the first and/or second recombinant AAV vector, and/or (b) reduces binding of the neutralizing antibody to an Fc receptor to do; and

(ii) administering to said subject an effective amount of a second recombinant AAV vector.

79. The method according to embodiment 78, wherein the first recombinant AAV and the second recombinant AAV are of the same serotype.

80. The method according to embodiment 78, wherein the first recombinant AAV and the second recombinant AAV are different serotypes.

81. The method according to any one of embodiments 78-80, wherein the neutralizing antibody is IgG, IgM, IgE or IgA.

82. The method according to embodiment 81, wherein the neutralizing antibody is IgG.

83. The embodiment, wherein the recombinant AAV vector is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh8, AAVrh10, AAVrh32.33, AAVrh74, avian AAV or bovine AAV vector. A method according to any one of Examples 76-82.

84. The method according to embodiment 83, wherein the AAV vector is a wild-type AAV vector.

85. The method according to embodiment 83, wherein the AAV vector is a mutant AAV vector.

86. The method according to any one of embodiments 78-85, wherein the recombinant AAV comprises a heterologous nucleic acid encoding a therapeutic protein or therapeutic RNA.

87. at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 99% of the antibody is degraded after administration of the composition , the method according to any one of embodiments 78-86.

88. The method according to any one of embodiments 78-87, wherein the composition comprises an antibody-degrading enzyme or fragment thereof.

89. A method according to any one of embodiments 78-87, wherein the composition comprises a vector comprising a polynucleotide encoding an antibody-degrading enzyme or fragment thereof.

90. A method according to embodiment 88 or 89, wherein the antibody-degrading enzyme or fragment thereof has cysteine protease activity.

91. The method according to any one of embodiments 78-90, wherein the antibody-degrading enzyme specifically cleaves IgG.

92. The method according to any one of embodiments 78-91, wherein the antibody-degrading enzyme or fragment thereof is derived from the genus Streptococcus.

93. The method according to any one of embodiments 78-92, wherein the antibody-degrading enzyme comprises an amino acid sequence that is at least 90% or at least 95% identical to the amino acid sequence of SEQ ID NO: 1.

94. The method according to embodiment 93, wherein the antibody-degrading enzyme comprises the amino acid sequence of SEQ ID NO: 1.

95. The method according to any one of embodiments 87-94, wherein the composition comprises a fusion protein comprising a first protein and a second protein, wherein the first protein is an antibody-degrading enzyme or fragment thereof.

96. The method according to embodiment 95, wherein the first protein and the second protein are separated by a linker.

97. A method according to embodiment 95 or 96, wherein the second protein is an IgG protease.

98. The method according to any one of embodiments 78-97, wherein about 0.1 mg/kg to about 100 mg/kg of the antibody-degrading enzyme or fragment thereof is administered to the subject.

99. The method according to any one of embodiments 78-98, wherein administration reduces binding of said antibody to Fc receptors.

100. The method according to any one of embodiments 78-99, wherein the composition is administered intravenously.

101. A method according to any one of embodiments 78-100, wherein the composition comprises a pharmaceutically acceptable carrier and/or diluent.

102. The method according to any one of embodiments 78-101, wherein the subject is a human.

103. an amount of an AAV vector effective in the subject, and (a) promotes degradation of a recombinant protein encoded by an antibody to the AAV vector or a heterologous nucleic acid; and/or (b) administering a composition that reduces binding of the antibody to an Fc receptor. .

104. The method according to embodiment 103, wherein the antibody is IgG.

105. A method according to embodiments 103 or 104, wherein said AAV vector is administered to the subject simultaneously with said composition.

106. The method according to embodiment 103 or 104, wherein said AAV vector is administered after administration of said composition to a subject.

107. A method according to embodiments 103 or 104, wherein said AAV vector is administered prior to administration of said composition to a subject.

108. The method according to embodiment 107, further comprising administering at least one second AAV vector comprising a second heterologous nucleic acid.

109. The method according to embodiment 108, wherein the AAV vector and said second AAV vector comprise an AAV capsid protein of the same serotype.

110. The method according to embodiment 108, wherein the AAV vector and the second AAV vector comprise AAV capsid proteins of different serotypes.

111. The method according to any one of embodiments 102-110, wherein the composition further comprises a pharmaceutically acceptable carrier and/or diluent.

112. The method according to any one of embodiments 102-111, wherein the composition promotes degradation of the antibody.

113. The embodiment, wherein the level of the antibody in the subject's body is reduced to a level in the range of from about 95% to about 0.01% relative to the antibody level in a control subject, the control subject being administered the AAV vector but not administered the composition. Method according to 112.

114. A method according to embodiment 112 or 113, wherein the composition comprises an antibody-degrading enzyme or fragment thereof.

115. A method according to embodiment 112 or 113, wherein the composition comprises a vector comprising a polynucleotide encoding an antibody-degrading enzyme or fragment thereof.

116. A method according to embodiment 114 or 115, wherein the antibody-degrading enzyme or fragment thereof comprises an IgG cysteine protease activity.

117. The method according to any one of embodiments 114-116, wherein the antibody-degrading enzyme or fragment thereof is derived from the genus Streptococcus.

118. The method according to any one of embodiments 114-117, wherein the antibody-degrading enzyme comprises an amino acid sequence that is at least 50% identical to the amino acid sequence of SEQ ID NO: 1.

119. The method according to any one of embodiments 114-118, wherein the antibody-degrading enzyme comprises the amino acid sequence of SEQ ID NO: 1.

120. The composition comprises an antibody-degrading enzyme or fragment thereof; and a fusion protein comprising a second protein.

121. The method according to embodiment 120, wherein the second protein is an IgG protease.

122. A method according to any one of embodiments 114-121, wherein the subject is administered about 0.1 mg/kg to about 100 mg/kg of the antibody-degrading enzyme or fragment thereof.

123. The method according to any one of embodiments 108-122, wherein the subject is a human.

124. A method according to embodiment 102, wherein the composition reduces binding of the antibody to an Fc receptor.

It should be understood that the embodiments described below as well as the above description are for illustrative purposes only and are not intended to limit the scope of the present invention. Other aspects, advantages and modifications within the scope of the present invention will be apparent to those skilled in the art to which this invention pertains.

Example

Example 1: recombination IdeZ ( rIdeZ )of cloning , expression and purification

The IdeZ coding sequence was cloned into pGEX-6P-3 vector using BamHI and SalI restriction enzyme sites to construct an IdeZ fusion (GST-IdeZ) with GST attached to the N-terminus ( FIG. 1A ). Expression of GST-IdeZ was controlled under the lac operon, and production was induced by IPTG addition. IdeZ protein was purified using glutathione sepharose and eluted with excess glutathione. Expression and purification were monitored using SDS-PAGE ( FIG. 1B ). Recombinant IdeZ was quantified using BSA as standard ^{in Biorad Imagelab™ software.}

Example 2: IdeZ is recombinant mouse IgG and from a plurality of species derived serum IgG cut the

To determine whether rIdeZ was active in vitro, mouse, primate and human serum samples were treated with recombinant GST-IdeZ (1 μg) at 37° C. for 3 hours. As shown in Figure 6 , GST-IdeZ cleaved IgG was present in human and primate serum.

In a separate experiment, recombinant mouse IgG (40 μg) was incubated with rIdeZ (NEB P0770S, 160 unit) for 2 hours at 37°C. The reaction was analyzed by SDS-PAGE under non-reducing conditions and stained with Coomassie blue ( FIG. 2A ). In the presence of IdeZ, recombinant mouse IgG was cleaved into several bands as indicated by asterisks. Arrows point to the IdeZ protein.

As an additional experiment, serum samples from mice, primates and humans were treated with recombinant IdeZ (NEB P0770S, 320 units) for 3 hours at 37°C either untreated (-) or treated (+). The reaction was diluted 1:10 and analyzed by SDS-PAGE under reducing conditions. The gel was then stained with Coomassie Blue. As shown in Figure 2B, IgG present in the serum was cleaved by IdeZ. In FIG. 2B , the bottom of the gel appears to be overexposed of the part of the gel containing the IgG heavy chain cleavage product (˜31 kDa). IdeZ also cleaved IgG in serum samples from other human patients (donors 1-5) ( FIG. 2D ). In a similar experiment, it was also observed that IdeZ was able to cleave IgG from dog serum ( FIG. 2C ). In sum, these data demonstrate that IdeZ is capable of cleaving IgG in the serum of several species.

Example 3: IdeZ is human IVIG in vitro and in vivo cut

Human intravenous immunoglobulin (IVIG) was incubated with GST-IdeZ (1 μg) or IdeZ (NEB P0770S or Genscript) for 2 hours at 37°C. The reaction was analyzed by SDS-PAGE under reducing conditions and stained with Coomassie Blue. As shown in Figure 3B , IdeZ cleaved human IVIG in vitro.

Mice were injected intraperitoneally with 8 mg of human IVIG. After 24 hours, the same mice were intravenously injected with PBS (-) or recombinant IdeZ (2.5 mg/kg) (+). At 72 hours after IVIG injection, blood samples were collected and then analyzed by SDS-PAGE under reducing conditions and immunoblotting was performed. IVIG was probed with goat anti-humIgG Alexa Fluor 647 (1:10,000). In the presence of IdeZ, human IVIG was cleaved into several small cleavage products as indicated by asterisks ( FIG. 3A ). These data show that IdeZ cleaves human IVIG in vivo.

In a similar experiment, mice were injected intraperitoneally with 8 mg of human IVIG. After 24 hours, the same mice were intravenously injected with PBS (-) or recombinant GST-IdeZ (2.5 mg/kg) (+). Blood samples were drawn before injection and at 24, 48 and 72 hours after IVIG injection. As shown in FIG. 7 , IdeZ cleaved human IVIG within 24 h, and the cleavage level continued to increase up to 72 h (3 days).

Example 4: GST - IdeZ medium IVIG Dose analysis for amputation

A dose analysis for GST-IdeZ mediated IVIG cleavage was also performed. In this experiment, mice were injected intraperitoneally with 8 mg of human IVIG. After 24 hours, the same mouse was intravenously injected with PBS (-) or recombinant GST-IdeZ (0.25 mg/kg) (+). As shown in Figures 8A-8B , cleavage was dose dependent. At the highest dose (2.5 mg/kg), almost all IVIG was cleaved in the sample, as confirmed by the band size variation.

The cleavage site of IdeZ is in the hinge region of human immunoglobulins. To verify that IdeZ cleaved IVIG, serum samples of mouse PBS(-) or 1 mg/kg IdeZ(+) were run on SDS-PAGE and then probed with anti-Fab or anti-Fc antibody. As shown in FIG. 9 , the Fab band size was changed due to the IdeZ treatment (about 250 -> about 150 kDa), which means that it was cleaved. In the IdeZ-treated sample, an Fc band was observed at about 50 kDa, indicating that this domain was separated from the Fab.

A neutralization profile of AAV8-Luc using human IVIG was also constructed. GST-IdeZ (1 μg) treated or untreated human IVIG was serially diluted 2-fold from 1:1000 to 1:102,400, incubated with AAV8-Luc and then administered to cells during culture (100,000 vg/cell). . As confirmed by the graph in FIG. 10 , the neutralization of AAV8-Luc was reduced in the presence of GST-IdeZ.

Example 5: IdeZ is IVIG in treated mice AAV8 -Luc liver transduction save

Mice were injected intraperitoneally with 8 mg of human IVIG. 24 hours later, PBS or recombinant IdeZ (2.5 mg/kg) and AAV8-Luc (5 x 10 ¹² vg/kg) were intravenously injected into the same mouse. The expression level of the luciferase transgene in the liver was analyzed 4 weeks after injection. Luciferase expression levels were normalized to the concentration of total tissue protein and expressed in relative luminous units per gram of liver tissue (RLU). All experiments were performed in 3 sets. * p <0.05.; LOD = limit of detection.

As shown in FIG. 4 , a decrease in the level of liver AAV8-Luc transduction was observed in IVIG-treated mice. However, when IVIG-treated mice were co-injected with IdeZ, AAV8-Luc liver transduction was observed at a level comparable to that of PBS-treated mice.

AAV8-Luc copy number was calculated in liver samples from mice. 11, the number of AAV-Luc copies per cell was higher in the samples of AAV8-Luc and IdeZ co-injected IVIG treated mice. Liver transduction was very low in IdeZ untreated mice.

In summary, these data show that IdeZ reduces AAV neutralization by IVIG and promotes liver transduction of AAV8-Luc.

Example 6: IdeZ is IVIG in treated mice AAV9 -Luc rescues liver and cardiac transduction

Mice were injected intraperitoneally with 8 mg of human IVIG. After 72 hours, the same mouse was intravenously injected with PBS or recombinant GST-IdeZ (2.5 mg/kg). 72 hours after IdeZ treatment, mice were treated with AAV9-Luc (2 x 10 ¹¹ vg/mouse). The expression level of the luciferase transgene was analyzed in the liver and heart at 4 weeks post-injection. Luciferase expression levels were normalized to the concentration of total tissue protein and expressed in relative luminous units per gram of liver tissue (RLU).

For IVIG-treated male and female mice, decreased levels of AAV9-Luc transduction were observed in liver ( FIG. 12A , 12C ) and heart ( FIG. 12B , 12D ). However, in IVIG-treated mice co-injected with GST-IdeZ, AAV9-Luc liver and cardiac transduction was observed at comparable levels to PBS-treated mice.

In summary, these data show that IdeZ negates IVIG-mediated neutralization of AAV and promotes AAV9-Luc liver and cardiac transduction (L.O.D = limit of detection).

Example 7: IdeZ is In mice treated with patient serum AAV9 -Luc liver and cardiac transduction improve

Serum samples obtained from 18 human patients were tested for their ability to neutralize AAV9 transduction in liver and heart.

Two mice per human serum sample were used in the experiment, both of which were injected intraperitoneally with 100 μl of human patient serum. Then, after 72 hours, the mice were intravenously injected with PBS or recombinant GST-IdeZ (2.5 mg/kg). 72 hours after IdeZ treatment, mice ^{were intravenously injected with AAV9-Luc (2 x 10 11} vg/mouse).

The level of transduction into liver and heart was analyzed 4 weeks after injection. Transduction levels were normalized to control mice injected with AAV9-Luc (2×10 ^{11 vg/mouse) without serum treatment.}

As shown in Figures 13A and 13B , differential transduction levels were observed in mice treated with human patient serum. In mice treated with patient serum with strong neutralizing properties, an increase in transduction in liver ( FIG. 13A ) and heart ( FIG. 13B ) was observed upon co-injection of GST-IdeZ.

In summary, these data show that IdeZ antagonizes patient serum-mediated neutralization of AAV and promotes transduction of AAV9-Luc into liver and heart.

Those skilled in the art will readily appreciate that the subject matter of the present invention is seamlessly adapted to achieve the objectives and advantages stated and implied. The content of the invention described herein describes and exemplifies preferred exemplary embodiments, and is not intended to limit the scope of the invention. A person skilled in the art may make modifications and other uses thereof, which are included in the spirit of the present invention as defined by the scope of the claims.

SEQUENCE LISTING <110> Duke University <120> COMPOSITIONS AND METHODS FOR EVADING HUMORAL IMMUNITY <130> DU6568PCT <150> US 62/914,682 <151> 2019-10-14 <150> US 62/797,495 <151> 2019-01-28 <160> 52 <170> PatentIn version 3.5 <210> 1 <211> 349 <212> PRT <213> Streptococcus equi <400> 1 Met Lys Thr Ile Ala Tyr Pro Asn Lys Pro His Ser Leu Ser Ala Gly 1 5 10 15 Leu Leu Thr Ala Ile Ala Ile Phe Ser Leu Ala Ser Ser Asn Ile Thr 20 25 30 Tyr Ala Asp Asp Tyr Gln Arg Asn Ala Thr Glu Ala Tyr Ala Lys Glu 35 40 45 Val Pro His Gln Ile Thr Ser Val Trp Ser Lys Gly Val Thr Pro Leu 50 55 60 Thr Pro Glu Gln Phe Arg Tyr Asn Asn Glu Asp Val Ile His Ala Pro 65 70 75 80 Tyr Leu Ala His Gln Gly Trp Tyr Asp Ile Thr Lys Ala Phe Asp Gly 85 90 95 Lys Asp Asn Leu Leu Cys Gly Ala Ala Thr Ala Gly Asn Met Leu His 100 105 110 Trp Trp Phe Asp Gln Asn Lys Thr Glu Ile Glu Ala Tyr Leu Ser Lys 115 120 125 His Pro Glu Lys Gln Lys Ile Ile Phe Asn Asn Gln Glu Leu Phe Asp 130 135 140 Leu Lys Ala Ala Ile Asp Thr Lys Asp Ser Gln Thr Asn Ser Gln Leu 145 150 155 160 Phe Asn Tyr Phe Arg Asp Lys Ala Phe Pro Asn Leu Ser Ala Arg Gln 165 170 175 Leu Gly Val Met Pro Asp Leu Val Leu Asp Met Phe Ile Asn Gly Tyr 180 185 190 Tyr Leu Asn Val Phe Lys Thr Gln Ser Thr Asp Val Asn Arg Pro Tyr 195 200 205 Gln Asp Lys Asp Lys Arg Gly Gly Ile Phe Asp Ala Val Phe Thr Arg 210 215 220 Gly Asp Gln Thr Thr Leu Leu Thr Ala Arg His Asp Leu Lys Asn Lys 225 230 235 240 Gly Leu Asn Asp Ile Ser Thr Ile Ile Lys Gln Glu Leu Thr Glu Gly 245 250 255 Arg Ala Leu Ala Leu Ser His Thr Tyr Ala Asn Val Ser Ile Ser His 260 265 270 Val Ile Asn Leu Trp Gly Ala Asp Phe Asn Ala Glu Gly Asn Leu Glu 275 280 285 Ala Ile Tyr Val Thr Asp Ser Asp Ala Asn Ala Ser Ile Gly Met Lys 290 295 300 Lys Tyr Phe Val Gly Ile Asn Ala His Gly His Val Ala Ile Ser Ala 305 310 315 320 Lys Lys Ile Glu Gly Glu Asn Ile Gly Ala Gln Val Leu Gly Leu Phe 325 330 335 Thr Leu Ser Ser Gly Lys Asp Ile Trp Gln Lys Leu Ser 340 345 <210> 2 <211> 736 <212> PRT <213> Parvovirus adeno-associated virus <400> 2 Met Ala Ala Asp Gly Tyr Leu Pro Asp Trp Leu Glu Asp Asn Leu Ser 1 5 10 15 Glu Gly Ile Arg Glu Trp Trp Asp Leu Lys Pro Gly Ala Pro Lys Pro 20 25 30 Lys Ala Asn Gln Gln Lys Gln Asp Asp Gly Arg Gly Leu Val Leu Pro 35 40 45 Gly Tyr Lys Tyr Leu Gly Pro Phe Asn Gly Leu Asp Lys Gly Glu Pro 50 55 60 Val Asn Ala Ala Asp Ala Ala Ala Leu Glu His Asp Lys Ala Tyr Asp 65 70 75 80 Gln Gln Leu Lys Ala Gly Asp Asn Pro Tyr Leu Arg Tyr Asn His Ala 85 90 95 Asp Ala Glu Phe Gln Glu Arg Leu Gln Glu Asp Thr Ser Phe Gly Gly 100 105 110 Asn Leu Gly Arg Ala Val Phe Gln Ala Lys Lys Arg Val Leu Glu Pro 115 120 125 Leu Gly Leu Val Glu Glu Gly Ala Lys Thr Ala Pro Gly Lys Lys Arg 130 135 140 Pro Val Glu Gln Ser Pro Gln Glu Pro Asp Ser Ser Ser Gly Ile Gly 145 150 155 160 Lys Thr Gly Gln Gln Pro Ala Lys Lys Arg Leu Asn Phe Gly Gln Thr 165 170 175 Gly Asp Ser Glu Ser Val Pro Asp Pro Gln Pro Leu Gly Glu Pro Pro 180 185 190 Ala Thr Pro Ala Ala Val Gly Pro Thr Thr Met Ala Ser Gly Gly Gly 195 200 205 Ala Pro Met Ala Asp Asn Asn Glu Gly Ala Asp Gly Val Gly Asn Ala 210 215 220 Ser Gly Asn Trp His Cys Asp Ser Thr Trp Leu Gly Asp Arg Val Ile 225 230 235 240 Thr Thr Ser Thr Arg Thr Trp Ala Leu Pro Thr Tyr Asn Asn His Leu 245 250 255 Tyr Lys Gln Ile Ser Ser Ala Ser Thr Gly Ala Ser Asn Asp Asn His 260 265 270 Tyr Phe Gly Tyr Ser Thr Pro Trp Gly Tyr Phe Asp Phe Asn Arg Phe 275 280 285 His Cys His Phe Ser Pro Arg Asp Trp Gln Arg Leu Ile Asn Asn Asn 290 295 300 Trp Gly Phe Arg Pro Lys Arg Leu Asn Phe Lys Leu Phe Asn Ile Gln 305 310 315 320 Val Lys Glu Val Thr Thr Asn Asp Gly Val Thr Thr Ile Ala Asn Asn 325 330 335 Leu Thr Ser Thr Val Gln Val Phe Ser Asp Ser Glu Tyr Gln Leu Pro 340 345 350 Tyr Val Leu Gly Ser Ala His Gln Gly Cys Leu Pro Pro Phe Pro Ala 355 360 365 Asp Val Phe Met Ile Pro Gln Tyr Gly Tyr Leu Thr Leu Asn Asn Gly 370 375 380 Ser Gln Ala Val Gly Arg Ser Ser Phe Tyr Cys Leu Glu Tyr Phe Pro 385 390 395 400 Ser Gln Met Leu Arg Thr Gly Asn Asn Phe Thr Phe Ser Tyr Thr Phe 405 410 415 Glu Glu Val Pro Phe His Ser Ser Tyr Ala His Ser Gln Ser Leu Asp 420 425 430 Arg Leu Met Asn Pro Leu Ile Asp Gln Tyr Leu Tyr Tyr Leu Asn Arg 435 440 445 Thr Gln Asn Gln Ser Gly Ser Ala Gln Asn Lys Asp Leu Leu Phe Ser 450 455 460 Arg Gly Ser Pro Ala Gly Met Ser Val Gln Pro Lys Asn Trp Leu Pro 465 470 475 480 Gly Pro Cys Tyr Arg Gln Gln Arg Val Ser Lys Thr Lys Thr Asp Asn 485 490 495 Asn Asn Ser Asn Phe Thr Trp Thr Gly Ala Ser Lys Tyr Asn Leu Asn 500 505 510 Gly Arg Glu Ser Ile Ile Asn Pro Gly Thr Ala Met Ala Ser His Lys 515 520 525 Asp Asp Glu Asp Lys Phe Phe Pro Met Ser Gly Val Met Ile Phe Gly 530 535 540 Lys Glu Ser Ala Gly Ala Ser Asn Thr Ala Leu Asp Asn Val Met Ile 545 550 555 560 Thr Asp Glu Glu Glu Ile Lys Ala Thr Asn Pro Val Ala Thr Glu Arg 565 570 575 Phe Gly Thr Val Ala Val Asn Phe Gln Ser Ser Ser Thr Asp Pro Ala 580 585 590 Thr Gly Asp Val His Ala Met Gly Ala Leu Pro Gly Met Val Trp Gln 595 600 605 Asp Arg Asp Val Tyr Leu Gln Gly Pro Ile Trp Ala Lys Ile Pro His 610 615 620 Thr Asp Gly His Phe His Pro Ser Pro Leu Met Gly Gly Phe Gly Leu 625 630 635 640 Lys Asn Pro Pro Gln Ile Leu Ile Lys Asn Thr Pro Val Pro Ala 645 650 655 Asn Pro Pro Ala Glu Phe Ser Ala Thr Lys Phe Ala Ser Phe Ile Thr 660 665 670 Gln Tyr Ser Thr Gly Gln Val Ser Val Glu Ile Glu Trp Glu Leu Gln 675 680 685 Lys Glu Asn Ser Lys Arg Trp Asn Pro Glu Val Gln Tyr Thr Ser Asn 690 695 700 Tyr Ala Lys Ser Ala Asn Val Asp Phe Thr Val Asp Asn Asn Gly Leu 705 710 715 720 Tyr Thr Glu Pro Arg Pro Ile Gly Thr Arg Tyr Leu Thr Arg Pro Leu 725 730 735 <210> 3 <211> 735 <212> PRT <213> Parvovirus adeno-associated virus <400> 3 Met Ala Ala Asp Gly Tyr Leu Pro Asp Trp Leu Glu Asp Thr Leu Ser 1 5 10 15 Glu Gly Ile Arg Gln Trp Trp Lys Leu Lys Pro Gly Pro Pro Pro Pro 20 25 30 Lys Pro Ala Glu Arg His Lys Asp Asp Ser Arg Gly Leu Val Leu Pro 35 40 45 Gly Tyr Lys Tyr Leu Gly Pro Phe Asn Gly Leu Asp Lys Gly Glu Pro 50 55 60 Val Asn Glu Ala Asp Ala Ala Ala Leu Glu His Asp Lys Ala Tyr Asp 65 70 75 80 Arg Gln Leu Asp Ser Gly Asp Asn Pro Tyr Leu Lys Tyr Asn His Ala 85 90 95 Asp Ala Glu Phe Gln Glu Arg Leu Lys Glu Asp Thr Ser Phe Gly Gly 100 105 110 Asn Leu Gly Arg Ala Val Phe Gln Ala Lys Lys Arg Val Leu Glu Pro 115 120 125 Leu Gly Leu Val Glu Glu Pro Val Lys Thr Ala Pro Gly Lys Lys Arg 130 135 140 Pro Val Glu His Ser Pro Val Glu Pro Asp Ser Ser Ser Gly Thr Gly 145 150 155 160 Lys Ala Gly Gln Gln Pro Ala Arg Lys Arg Leu Asn Phe Gly Gln Thr 165 170 175 Gly Asp Ala Asp Ser Val Pro Asp Pro Gln Pro Leu Gly Gln Pro Pro 180 185 190 Ala Ala Pro Ser Gly Leu Gly Thr Asn Thr Met Ala Thr Gly Ser Gly 195 200 205 Ala Pro Met Ala Asp Asn Asn Glu Gly Ala Asp Gly Val Gly Asn Ser 210 215 220 Ser Gly Asn Trp His Cys Asp Ser Thr Trp Met Gly Asp Arg Val Ile 225 230 235 240 Thr Thr Ser Thr Arg Thr Trp Ala Leu Pro Thr Tyr Asn Asn His Leu 245 250 255 Tyr Lys Gln Ile Ser Ser Gln Ser Gly Ala Ser Asn Asp Asn His Tyr 260 265 270 Phe Gly Tyr Ser Thr Pro Trp Gly Tyr Phe Asp Phe Asn Arg Phe His 275 280 285 Cys His Phe Ser Pro Arg Asp Trp Gln Arg Leu Ile Asn Asn Asn Trp 290 295 300 Gly Phe Arg Pro Lys Arg Leu Asn Phe Lys Leu Phe Asn Ile Gln Val 305 310 315 320 Lys Glu Val Thr Gln Asn Asp Gly Thr Thr Thr Ile Ala Asn Asn Leu 325 330 335 Thr Ser Thr Val Gln Val Phe Thr Asp Ser Glu Tyr Gln Leu Pro Tyr 340 345 350 Val Leu Gly Ser Ala His Gln Gly Cys Leu Pro Pro Phe Pro Ala Asp 355 360 365 Val Phe Met Val Pro Gln Tyr Gly Tyr Leu Thr Leu Asn Asn Gly Ser 370 375 380 Gln Ala Val Gly Arg Ser Ser Phe Tyr Cys Leu Glu Tyr Phe Pro Ser 385 390 395 400 Gln Met Leu Arg Thr Gly Asn Asn Phe Thr Phe Ser Tyr Thr Phe Glu 405 410 415 Asp Val Pro Phe His Ser Ser Tyr Ala His Ser Gln Ser Leu Asp Arg 420 425 430 Leu Met Asn Pro Leu Ile Asp Gln Tyr Leu Tyr Tyr Leu Ser Arg Thr 435 440 445 Asn Thr Pro Ser Gly Thr Thr Thr Gln Ser Arg Leu Gln Phe Ser Gln 450 455 460 Ala Gly Ala Ser Asp Ile Arg Asp Gln Ser Arg Asn Trp Leu Pro Gly 465 470 475 480 Pro Cys Tyr Arg Gln Gln Arg Val Ser Lys Thr Ser Ala Asp Asn Asn 485 490 495 Asn Ser Glu Tyr Ser Trp Thr Gly Ala Thr Lys Tyr His Leu Asn Gly 500 505 510 Arg Asp Ser Leu Val Asn Pro Gly Pro Ala Met Ala Ser His Lys Asp 515 520 525 Asp Glu Glu Lys Phe Phe Pro Gln Ser Gly Val Leu Ile Phe Gly Lys 530 535 540 Gln Gly Ser Glu Lys Thr Asn Val Asp Ile Glu Lys Val Met Ile Thr 545 550 555 560 Asp Glu Glu Glu Ile Arg Thr Thr Asn Pro Val Ala Thr Glu Gln Tyr 565 570 575 Gly Ser Val Ser Thr Asn Leu Gln Arg Gly Asn Arg Gln Ala Ala Thr 580 585 590 Ala Asp Val Asn Thr Gln Gly Val Leu Pro Gly Met Val Trp Gln Asp 595 600 605 Arg Asp Val Tyr Leu Gln Gly Pro Ile Trp Ala Lys Ile Pro His Thr 610 615 620 Asp Gly His Phe His Pro Ser Pro Leu Met Gly Gly Phe Gly Leu Lys 625 630 635 640 His Pro Pro Pro Gln Ile Leu Ile Lys Asn Thr Pro Val Pro Ala Asn 645 650 655 Pro Ser Thr Thr Phe Ser Ala Ala Lys Phe Ala Ser Phe Ile Thr Gln 660 665 670 Tyr Ser Thr Gly Gln Val Ser Val Glu Ile Glu Trp Glu Leu Gln Lys 675 680 685 Glu Asn Ser Lys Arg Trp Asn Pro Glu Ile Gln Tyr Thr Ser Asn Tyr 690 695 700 Asn Lys Ser Val Asn Val Asp Phe Thr Val Asp Thr Asn Gly Val Tyr 705 710 715 720 Ser Glu Pro Arg Pro Ile Gly Thr Arg Tyr Leu Thr Arg Asn Leu 725 730 735 <210> 4 <211> 736 <212> PRT <213> Parvovirus adeno-associated virus <400> 4 Met Ala Ala Asp Gly Tyr Leu Pro Asp Trp Leu Glu Asp Asn Leu Ser 1 5 10 15 Glu Gly Ile Arg Glu Trp Trp Ala Leu Lys Pro Gly Val Pro Gln Pro 20 25 30 Lys Ala Asn Gln Gln His Gln Asp Asn Arg Arg Gly Leu Val Leu Pro 35 40 45 Gly Tyr Lys Tyr Leu Gly Pro Gly Asn Gly Leu Asp Lys Gly Glu Pro 50 55 60 Val Asn Glu Ala Asp Ala Ala Ala Leu Glu His Asp Lys Ala Tyr Asp 65 70 75 80 Gln Gln Leu Lys Ala Gly Asp Asn Pro Tyr Leu Lys Tyr Asn His Ala 85 90 95 Asp Ala Glu Phe Gln Glu Arg Leu Gln Glu Asp Thr Ser Phe Gly Gly 100 105 110 Asn Leu Gly Arg Ala Val Phe Gln Ala Lys Lys Arg Ile Leu Glu Pro 115 120 125 Leu Gly Leu Val Glu Glu Ala Ala Lys Thr Ala Pro Gly Lys Lys Arg 130 135 140 Pro Val Asp Gln Ser Pro Gln Glu Pro Asp Ser Ser Ser Gly Val Gly 145 150 155 160 Lys Ser Gly Lys Gln Pro Ala Arg Lys Arg Leu Asn Phe Gly Gln Thr 165 170 175 Gly Asp Ser Glu Ser Val Pro Asp Pro Gln Pro Leu Gly Glu Pro Pro 180 185 190 Ala Ala Pro Thr Ser Leu Gly Ser Asn Thr Met Ala Ser Gly Gly Gly 195 200 205 Ala Pro Met Ala Asp Asn Asn Glu Gly Ala Asp Gly Val Gly Asn Ser 210 215 220 Ser Gly Asn Trp His Cys Asp Ser Gln Trp Leu Gly Asp Arg Val Ile 225 230 235 240 Thr Thr Ser Thr Arg Thr Trp Ala Leu Pro Thr Tyr Asn Asn His Leu 245 250 255 Tyr Lys Gln Ile Ser Ser Gln Ser Gly Ala Ser Asn Asp Asn His Tyr 260 265 270 Phe Gly Tyr Ser Thr Pro Trp Gly Tyr Phe Asp Phe Asn Arg Phe His 275 280 285 Cys His Phe Ser Pro Arg Asp Trp Gln Arg Leu Ile Asn Asn Asn Trp 290 295 300 Gly Phe Arg Pro Lys Lys Leu Ser Phe Lys Leu Phe Asn Ile Gln Val 305 310 315 320 Lys Glu Val Thr Gln Asn Asp Gly Thr Thr Thr Ile Ala Asn Asn Leu 325 330 335 Thr Ser Thr Val Gln Val Phe Thr Asp Ser Glu Tyr Gln Leu Pro Tyr 340 345 350 Val Leu Gly Ser Ala His Gln Gly Cys Leu Pro Pro Phe Pro Ala Asp 355 360 365 Val Phe Met Val Pro Gln Tyr Gly Tyr Leu Thr Leu Asn Asn Gly Ser 370 375 380 Gln Ala Val Gly Arg Ser Ser Phe Tyr Cys Leu Glu Tyr Phe Pro Ser 385 390 395 400 Gln Met Leu Arg Thr Gly Asn Asn Phe Gln Phe Ser Tyr Thr Phe Glu 405 410 415 Asp Val Pro Phe His Ser Ser Tyr Ala His Ser Gln Ser Leu Asp Arg 420 425 430 Leu Met Asn Pro Leu Ile Asp Gln Tyr Leu Tyr Tyr Leu Asn Arg Thr 435 440 445 Gln Gly Thr Thr Ser Gly Thr Thr Asn Gln Ser Arg Leu Leu Phe Ser 450 455 460 Gln Ala Gly Pro Gln Ser Met Ser Leu Gln Ala Arg Asn Trp Leu Pro 465 470 475 480 Gly Pro Cys Tyr Arg Gln Gln Arg Leu Ser Lys Thr Ala Asn Asp Asn 485 490 495 Asn Asn Ser Asn Phe Pro Trp Thr Ala Ala Ser Lys Tyr His Leu Asn 500 505 510 Gly Arg Asp Ser Leu Val Asn Pro Gly Pro Ala Met Ala Ser His Lys 515 520 525 Asp Asp Glu Glu Lys Phe Phe Pro Met His Gly Asn Leu Ile Phe Gly 530 535 540 Lys Glu Gly Thr Thr Ala Ser Asn Ala Glu Leu Asp Asn Val Met Ile 545 550 555 560 Thr Asp Glu Glu Glu Ile Arg Thr Thr Asn Pro Val Ala Thr Glu Gln 565 570 575 Tyr Gly Thr Val Ala Asn Asn Leu Gln Ser Ser Asn Thr Ala Pro Thr 580 585 590 Thr Arg Thr Val Asn Asp Gln Gly Ala Leu Pro Gly Met Val Trp Gln 595 600 605 Asp Arg Asp Val Tyr Leu Gln Gly Pro Ile Trp Ala Lys Ile Pro His 610 615 620 Thr Asp Gly His Phe His Pro Ser Pro Leu Met Gly Gly Phe Gly Leu 625 630 635 640 Lys His Pro Pro Pro Gln Ile Met Ile Lys Asn Thr Pro Val Pro Ala 645 650 655 Asn Pro Pro Thr Thr Phe Ser Pro Ala Lys Phe Ala Ser Phe Ile Thr 660 665 670 Gln Tyr Ser Thr Gly Gln Val Ser Val Glu Ile Glu Trp Glu Leu Gln 675 680 685 Lys Glu Asn Ser Lys Arg Trp Asn Pro Glu Ile Gln Tyr Thr Ser Asn 690 695 700 Tyr Asn Lys Ser Val Asn Val Asp Phe Thr Val Asp Thr Asn Gly Val 705 710 715 720 Tyr Ser Glu Pro Arg Pro Ile Gly Thr Arg Tyr Leu Thr Arg Asn Leu 725 730 735 <210> 5 <211> 734 <212> PRT <213> Parvovirus adeno-associated virus <400> 5 Met Thr Asp Gly Tyr Leu Pro Asp Trp Leu Glu Asp Asn Leu Ser Glu 1 5 10 15 Gly Val Arg Glu Trp Trp Ala Leu Gln Pro Gly Ala Pro Lys Pro Lys 20 25 30 Ala Asn Gln Gln His Gln Asp Asn Ala Arg Gly Leu Val Leu Pro Gly 35 40 45 Tyr Lys Tyr Leu Gly Pro Gly Asn Gly Leu Asp Lys Gly Glu Pro Val 50 55 60 Asn Ala Ala Asp Ala Ala Ala Leu Glu His Asp Lys Ala Tyr Asp Gln 65 70 75 80 Gln Leu Lys Ala Gly Asp Asn Pro Tyr Leu Lys Tyr Asn His Ala Asp 85 90 95 Ala Glu Phe Gln Gln Arg Leu Gln Gly Asp Thr Ser Phe Gly Gly Asn 100 105 110 Leu Gly Arg Ala Val Phe Gln Ala Lys Lys Arg Val Leu Glu Pro Leu 115 120 125 Gly Leu Val Glu Gln Ala Gly Glu Thr Ala Pro Gly Lys Lys Arg Pro 130 135 140 Leu Ile Glu Ser Pro Gln Gln Pro Asp Ser Ser Thr Gly Ile Gly Lys 145 150 155 160 Lys Gly Lys Gln Pro Ala Lys Lys Lys Leu Val Phe Glu Asp Glu Thr 165 170 175 Gly Ala Gly Asp Gly Pro Pro Glu Gly Ser Thr Ser Gly Ala Met Ser 180 185 190 Asp Asp Ser Glu Met Arg Ala Ala Ala Gly Gly Ala Ala Val Glu Gly 195 200 205 Gly Gln Gly Ala Asp Gly Val Gly Asn Ala Ser Gly Asp Trp His Cys 210 215 220 Asp Ser Thr Trp Ser Glu Gly His Val Thr Thr Thr Ser Thr Arg Thr 225 230 235 240 Trp Val Leu Pro Thr Tyr Asn Asn His Leu Tyr Lys Arg Leu Gly Glu 245 250 255 Ser Leu Gln Ser Asn Thr Tyr Asn Gly Phe Ser Thr Pro Trp Gly Tyr 260 265 270 Phe Asp Phe Asn Arg Phe His Cys His Phe Ser Pro Arg Asp Trp Gln 275 280 285 Arg Leu Ile Asn Asn Asn Trp Gly Met Arg Pro Lys Ala Met Arg Val 290 295 300 Lys Ile Phe Asn Ile Gln Val Lys Glu Val Thr Thr Ser Asn Gly Glu 305 310 315 320 Thr Thr Val Ala Asn Asn Leu Thr Ser Thr Val Gln Ile Phe Ala Asp 325 330 335 Ser Ser Tyr Glu Leu Pro Tyr Val Met Asp Ala Gly Gln Glu Gly Ser 340 345 350 Leu Pro Pro Phe Pro Asn Asp Val Phe Met Val Pro Gln Tyr Gly Tyr 355 360 365 Cys Gly Leu Val Thr Gly Asn Thr Ser Gln Gln Gln Thr Asp Arg Asn 370 375 380 Ala Phe Tyr Cys Leu Glu Tyr Phe Pro Ser Gln Met Leu Arg Thr Gly 385 390 395 400 Asn Asn Phe Glu Ile Thr Tyr Ser Phe Glu Lys Val Pro Phe His Ser 405 410 415 Met Tyr Ala His Ser Gln Ser Leu Asp Arg Leu Met Asn Pro Leu Ile 420 425 430 Asp Gln Tyr Leu Trp Gly Leu Gln Ser Thr Thr Thr Gly Thr Thr Leu 435 440 445 Asn Ala Gly Thr Ala Thr Thr Asn Phe Thr Lys Leu Arg Pro Thr Asn 450 455 460 Phe Ser Asn Phe Lys Lys Asn Trp Leu Pro Gly Pro Ser Ile Lys Gln 465 470 475 480 Gln Gly Phe Ser Lys Thr Ala Asn Gln Asn Tyr Lys Ile Pro Ala Thr 485 490 495 Gly Ser Asp Ser Leu Ile Lys Tyr Glu Thr His Ser Thr Leu Asp Gly 500 505 510 Arg Trp Ser Ala Leu Thr Pro Gly Pro Pro Met Ala Thr Ala Gly Pro 515 520 525 Ala Asp Ser Lys Phe Ser Asn Ser Gln Leu Ile Phe Ala Gly Pro Lys 530 535 540 Gln Asn Gly Asn Thr Ala Thr Val Pro Gly Thr Leu Ile Phe Thr Ser 545 550 555 560 Glu Glu Glu Leu Ala Ala Thr Asn Ala Thr Asp Thr Asp Met Trp Gly 565 570 575 Asn Leu Pro Gly Gly Asp Gln Ser Asn Ser Asn Leu Pro Thr Val Asp 580 585 590 Arg Leu Thr Ala Leu Gly Ala Val Pro Gly Met Val Trp Gln Asn Arg 595 600 605 Asp Ile Tyr Tyr Gln Gly Pro Ile Trp Ala Lys Ile Pro His Thr Asp 610 615 620 Gly His Phe His Pro Ser Pro Leu Ile Gly Gly Phe Gly Leu Lys His 625 630 635 640 Pro Pro Pro Gln Ile Phe Ile Lys Asn Thr Pro Val Pro Ala Asn Pro 645 650 655 Ala Thr Thr Phe Ser Ser Thr Pro Val Asn Ser Phe Ile Thr Gln Tyr 660 665 670 Ser Thr Gly Gln Val Ser Val Gln Ile Asp Trp Glu Ile Gln Lys Glu 675 680 685 Arg Ser Lys Arg Trp Asn Pro Glu Val Gln Phe Thr Ser Asn Tyr Gly 690 695 700 Gln Gln Asn Ser Leu Leu Trp Ala Pro Asp Ala Ala Gly Lys Tyr Thr 705 710 715 720 Glu Pro Arg Ala Ile Gly Thr Arg Tyr Leu Thr His His Leu 725 730 <210> 6 <211> 724 <212> PRT <213> Parvovirus adeno-associated virus <400> 6 Met Ser Phe Val Asp His Pro Asp Trp Leu Glu Glu Val Gly Glu 1 5 10 15 Gly Leu Arg Glu Phe Leu Gly Leu Glu Ala Gly Pro Pro Lys Pro Lys 20 25 30 Pro Asn Gln Gln His Gln Asp Gln Ala Arg Gly Leu Val Leu Pro Gly 35 40 45 Tyr Asn Tyr Leu Gly Pro Gly Asn Gly Leu Asp Arg Gly Glu Pro Val 50 55 60 Asn Arg Ala Asp Glu Val Ala Arg Glu His Asp Ile Ser Tyr Asn Glu 65 70 75 80 Gln Leu Glu Ala Gly Asp Asn Pro Tyr Leu Lys Tyr Asn His Ala Asp 85 90 95 Ala Glu Phe Gln Glu Lys Leu Ala Asp Asp Thr Ser Phe Gly Gly Asn 100 105 110 Leu Gly Lys Ala Val Phe Gln Ala Lys Lys Arg Val Leu Glu Pro Phe 115 120 125 Gly Leu Val Glu Glu Gly Ala Lys Thr Ala Pro Thr Gly Lys Arg Ile 130 135 140 Asp Asp His Phe Pro Lys Arg Lys Lys Ala Arg Thr Glu Glu Asp Ser 145 150 155 160 Lys Pro Ser Thr Ser Ser Asp Ala Glu Ala Gly Pro Ser Gly Ser Gln 165 170 175 Gln Leu Gln Ile Pro Ala Gln Pro Ala Ser Ser Leu Gly Ala Asp Thr 180 185 190 Met Ser Ala Gly Gly Gly Gly Pro Leu Gly Asp Asn Asn Gln Gly Ala 195 200 205 Asp Gly Val Gly Asn Ala Ser Gly Asp Trp His Cys Asp Ser Thr Trp 210 215 220 Met Gly Asp Arg Val Val Thr Lys Ser Thr Arg Thr Trp Val Leu Pro 225 230 235 240 Ser Tyr Asn Asn His Gln Tyr Arg Glu Ile Lys Ser Gly Ser Val Asp 245 250 255 Gly Ser Asn Ala Asn Ala Tyr Phe Gly Tyr Ser Thr Pro Trp Gly Tyr 260 265 270 Phe Asp Phe Asn Arg Phe His Ser His Trp Ser Pro Arg Asp Trp Gln 275 280 285 Arg Leu Ile Asn Asn Tyr Trp Gly Phe Arg Pro Arg Ser Leu Arg Val 290 295 300 Lys Ile Phe Asn Ile Gln Val Lys Glu Val Thr Val Gln Asp Ser Thr 305 310 315 320 Thr Thr Ile Ala Asn Asn Leu Thr Ser Thr Val Gln Val Phe Thr Asp 325 330 335 Asp Asp Tyr Gln Leu Pro Tyr Val Val Gly Asn Gly Thr Glu Gly Cys 340 345 350 Leu Pro Ala Phe Pro Pro Gln Val Phe Thr Leu Pro Gln Tyr Gly Tyr 355 360 365 Ala Thr Leu Asn Arg Asp Asn Thr Glu Asn Pro Thr Glu Arg Ser Ser 370 375 380 Phe Phe Cys Leu Glu Tyr Phe Pro Ser Lys Met Leu Arg Thr Gly Asn 385 390 395 400 Asn Phe Glu Phe Thr Tyr Asn Phe Glu Glu Val Pro Phe His Ser Ser 405 410 415 Phe Ala Pro Ser Gln Asn Leu Phe Lys Leu Ala Asn Pro Leu Val Asp 420 425 430 Gln Tyr Leu Tyr Arg Phe Val Ser Thr Asn Asn Thr Gly Gly Val Gln 435 440 445 Phe Asn Lys Asn Leu Ala Gly Arg Tyr Ala Asn Thr Tyr Lys Asn Trp 450 455 460 Phe Pro Gly Pro Met Gly Arg Thr Gln Gly Trp Asn Leu Gly Ser Gly 465 470 475 480 Val Asn Arg Ala Ser Val Ser Ala Phe Ala Thr Thr Asn Arg Met Glu 485 490 495 Leu Glu Gly Ala Ser Tyr Gln Val Pro Gln Pro Asn Gly Met Thr 500 505 510 Asn Asn Leu Gln Gly Ser Asn Thr Tyr Ala Leu Glu Asn Thr Met Ile 515 520 525 Phe Asn Ser Gln Pro Ala Asn Pro Gly Thr Thr Ala Thr Tyr Leu Glu 530 535 540 Gly Asn Met Leu Ile Thr Ser Glu Ser Glu Thr Gln Pro Val Asn Arg 545 550 555 560 Val Ala Tyr Asn Val Gly Gly Gln Met Ala Thr Asn Asn Gln Ser Ser 565 570 575 Thr Thr Ala Pro Ala Thr Gly Thr Tyr Asn Leu Gln Glu Ile Val Pro 580 585 590 Gly Ser Val Trp Met Glu Arg Asp Val Tyr Leu Gln Gly Pro Ile Trp 595 600 605 Ala Lys Ile Pro Glu Thr Gly Ala His Phe His Pro Ser Pro Ala Met 610 615 620 Gly Gly Phe Gly Leu Lys His Pro Pro Met Met Leu Ile Lys Asn 625 630 635 640 Thr Pro Val Pro Gly Asn Ile Thr Ser Phe Ser Asp Val Pro Val Ser 645 650 655 Ser Phe Ile Thr Gln Tyr Ser Thr Gly Gin Val Thr Val Glu Met Glu 660 665 670 Trp Glu Leu Lys Lys Glu Asn Ser Lys Arg Trp Asn Pro Glu Ile Gln 675 680 685 Tyr Thr Asn Asn Tyr Asn Asp Pro Gln Phe Val Asp Phe Ala Pro Asp 690 695 700 Ser Thr Gly Glu Tyr Arg Thr Thr Arg Pro Ile Gly Thr Arg Tyr Leu 705 710 715 720 Thr Arg Pro Leu <210> 7 <211> 736 <212> PRT <213> Parvovirus adeno-associated virus <400> 7 Met Ala Ala Asp Gly Tyr Leu Pro Asp Trp Leu Glu Asp Asn Leu Ser 1 5 10 15 Glu Gly Ile Arg Glu Trp Trp Asp Leu Lys Pro Gly Ala Pro Lys Pro 20 25 30 Lys Ala Asn Gln Gln Lys Gln Asp Asp Gly Arg Gly Leu Val Leu Pro 35 40 45 Gly Tyr Lys Tyr Leu Gly Pro Phe Asn Gly Leu Asp Lys Gly Glu Pro 50 55 60 Val Asn Ala Ala Asp Ala Ala Ala Leu Glu His Asp Lys Ala Tyr Asp 65 70 75 80 Gln Gln Leu Lys Ala Gly Asp Asn Pro Tyr Leu Arg Tyr Asn His Ala 85 90 95 Asp Ala Glu Phe Gln Glu Arg Leu Gln Glu Asp Thr Ser Phe Gly Gly 100 105 110 Asn Leu Gly Arg Ala Val Phe Gln Ala Lys Lys Arg Val Leu Glu Pro 115 120 125 Leu Gly Leu Val Glu Glu Gly Ala Lys Thr Ala Pro Gly Lys Lys Arg 130 135 140 Pro Val Glu Gln Ser Pro Gln Glu Pro Asp Ser Ser Ser Gly Ile Gly 145 150 155 160 Lys Thr Gly Gln Gln Pro Ala Lys Lys Arg Leu Asn Phe Gly Gln Thr 165 170 175 Gly Asp Ser Glu Ser Val Pro Asp Pro Gln Pro Leu Gly Glu Pro Pro 180 185 190 Ala Thr Pro Ala Ala Val Gly Pro Thr Thr Met Ala Ser Gly Gly Gly 195 200 205 Ala Pro Met Ala Asp Asn Asn Glu Gly Ala Asp Gly Val Gly Asn Ala 210 215 220 Ser Gly Asn Trp His Cys Asp Ser Thr Trp Leu Gly Asp Arg Val Ile 225 230 235 240 Thr Thr Ser Thr Arg Thr Trp Ala Leu Pro Thr Tyr Asn Asn His Leu 245 250 255 Tyr Lys Gln Ile Ser Ser Ala Ser Thr Gly Ala Ser Asn Asp Asn His 260 265 270 Tyr Phe Gly Tyr Ser Thr Pro Trp Gly Tyr Phe Asp Phe Asn Arg Phe 275 280 285 His Cys His Phe Ser Pro Arg Asp Trp Gln Arg Leu Ile Asn Asn Asn 290 295 300 Trp Gly Phe Arg Pro Lys Arg Leu Asn Phe Lys Leu Phe Asn Ile Gln 305 310 315 320 Val Lys Glu Val Thr Thr Asn Asp Gly Val Thr Thr Ile Ala Asn Asn 325 330 335 Leu Thr Ser Thr Val Gln Val Phe Ser Asp Ser Glu Tyr Gln Leu Pro 340 345 350 Tyr Val Leu Gly Ser Ala His Gln Gly Cys Leu Pro Pro Phe Pro Ala 355 360 365 Asp Val Phe Met Ile Pro Gln Tyr Gly Tyr Leu Thr Leu Asn Asn Gly 370 375 380 Ser Gln Ala Val Gly Arg Ser Ser Phe Tyr Cys Leu Glu Tyr Phe Pro 385 390 395 400 Ser Gln Met Leu Arg Thr Gly Asn Asn Phe Thr Phe Ser Tyr Thr Phe 405 410 415 Glu Asp Val Pro Phe His Ser Ser Tyr Ala His Ser Gln Ser Leu Asp 420 425 430 Arg Leu Met Asn Pro Leu Ile Asp Gln Tyr Leu Tyr Tyr Leu Asn Arg 435 440 445 Thr Gln Asn Gln Ser Gly Ser Ala Gln Asn Lys Asp Leu Leu Phe Ser 450 455 460 Arg Gly Ser Pro Ala Gly Met Ser Val Gln Pro Lys Asn Trp Leu Pro 465 470 475 480 Gly Pro Cys Tyr Arg Gln Gln Arg Val Ser Lys Thr Lys Thr Asp Asn 485 490 495 Asn Asn Ser Asn Phe Thr Trp Thr Gly Ala Ser Lys Tyr Asn Leu Asn 500 505 510 Gly Arg Glu Ser Ile Ile Asn Pro Gly Thr Ala Met Ala Ser His Lys 515 520 525 Asp Asp Lys Asp Lys Phe Phe Pro Met Ser Gly Val Met Ile Phe Gly 530 535 540 Lys Glu Ser Ala Gly Ala Ser Asn Thr Ala Leu Asp Asn Val Met Ile 545 550 555 560 Thr Asp Glu Glu Glu Ile Lys Ala Thr Asn Pro Val Ala Thr Glu Arg 565 570 575 Phe Gly Thr Val Ala Val Asn Leu Gln Ser Ser Ser Thr Asp Pro Ala 580 585 590 Thr Gly Asp Val His Val Met Gly Ala Leu Pro Gly Met Val Trp Gln 595 600 605 Asp Arg Asp Val Tyr Leu Gln Gly Pro Ile Trp Ala Lys Ile Pro His 610 615 620 Thr Asp Gly His Phe His Pro Ser Pro Leu Met Gly Gly Phe Gly Leu 625 630 635 640 Lys His Pro Pro Pro Gln Ile Leu Ile Lys Asn Thr Pro Val Pro Ala 645 650 655 Asn Pro Pro Ala Glu Phe Ser Ala Thr Lys Phe Ala Ser Phe Ile Thr 660 665 670 Gln Tyr Ser Thr Gly Gln Val Ser Val Glu Ile Glu Trp Glu Leu Gln 675 680 685 Lys Glu Asn Ser Lys Arg Trp Asn Pro Glu Val Gln Tyr Thr Ser Asn 690 695 700 Tyr Ala Lys Ser Ala Asn Val Asp Phe Thr Val Asp Asn Asn Gly Leu 705 710 715 720 Tyr Thr Glu Pro Arg Pro Ile Gly Thr Arg Tyr Leu Thr Arg Pro Leu 725 730 735 <210> 8 <211> 737 <212> PRT <213> Parvovirus adeno-associated virus <400> 8 Met Ala Ala Asp Gly Tyr Leu Pro Asp Trp Leu Glu Asp Asn Leu Ser 1 5 10 15 Glu Gly Ile Arg Glu Trp Trp Asp Leu Lys Pro Gly Ala Pro Lys Pro 20 25 30 Lys Ala Asn Gln Gln Lys Gln Asp Asn Gly Arg Gly Leu Val Leu Pro 35 40 45 Gly Tyr Lys Tyr Leu Gly Pro Phe Asn Gly Leu Asp Lys Gly Glu Pro 50 55 60 Val Asn Ala Ala Asp Ala Ala Ala Leu Glu His Asp Lys Ala Tyr Asp 65 70 75 80 Gln Gln Leu Lys Ala Gly Asp Asn Pro Tyr Leu Arg Tyr Asn His Ala 85 90 95 Asp Ala Glu Phe Gln Glu Arg Leu Gln Glu Asp Thr Ser Phe Gly Gly 100 105 110 Asn Leu Gly Arg Ala Val Phe Gln Ala Lys Lys Arg Val Leu Glu Pro 115 120 125 Leu Gly Leu Val Glu Glu Gly Ala Lys Thr Ala Pro Ala Lys Lys Arg 130 135 140 Pro Val Glu Pro Ser Pro Gln Arg Ser Pro Asp Ser Ser Thr Gly Ile 145 150 155 160 Gly Lys Lys Gly Gln Gln Pro Ala Arg Lys Arg Leu Asn Phe Gly Gln 165 170 175 Thr Gly Asp Ser Glu Ser Val Pro Asp Pro Gln Pro Leu Gly Glu Pro 180 185 190 Pro Ala Ala Pro Ser Ser Val Gly Ser Gly Thr Val Ala Ala Gly Gly 195 200 205 Gly Ala Pro Met Ala Asp Asn Asn Glu Gly Ala Asp Gly Val Gly Asn 210 215 220 Ala Ser Gly Asn Trp His Cys Asp Ser Thr Trp Leu Gly Asp Arg Val 225 230 235 240 Ile Thr Thr Ser Thr Arg Thr Trp Ala Leu Pro Thr Tyr Asn Asn His 245 250 255 Leu Tyr Lys Gln Ile Ser Ser Glu Thr Ala Gly Ser Thr Asn Asp Asn 260 265 270 Thr Tyr Phe Gly Tyr Ser Thr Pro Trp Gly Tyr Phe Asp Phe Asn Arg 275 280 285 Phe His Cys His Phe Ser Pro Arg Asp Trp Gln Arg Leu Ile Asn Asn 290 295 300 Asn Trp Gly Phe Arg Pro Lys Lys Leu Arg Phe Lys Leu Phe Asn Ile 305 310 315 320 Gln Val Lys Glu Val Thr Thr Asn Asp Gly Val Thr Thr Ile Ala Asn 325 330 335 Asn Leu Thr Ser Thr Ile Gln Val Phe Ser Asp Ser Glu Tyr Gln Leu 340 345 350 Pro Tyr Val Leu Gly Ser Ala His Gln Gly Cys Leu Pro Pro Phe Pro 355 360 365 Ala Asp Val Phe Met Ile Pro Gln Tyr Gly Tyr Leu Thr Leu Asn Asn 370 375 380 Gly Ser Gln Ser Val Gly Arg Ser Ser Phe Tyr Cys Leu Glu Tyr Phe 385 390 395 400 Pro Ser Gln Met Leu Arg Thr Gly Asn Asn Phe Glu Phe Ser Tyr Ser 405 410 415 Phe Glu Asp Val Pro Phe His Ser Ser Tyr Ala His Ser Gln Ser Leu 420 425 430 Asp Arg Leu Met Asn Pro Leu Ile Asp Gln Tyr Leu Tyr Tyr Leu Ala 435 440 445 Arg Thr Gln Ser Asn Pro Gly Gly Thr Ala Gly Asn Arg Glu Leu Gln 450 455 460 Phe Tyr Gln Gly Gly Pro Ser Thr Met Ala Glu Gln Ala Lys Asn Trp 465 470 475 480 Leu Pro Gly Pro Cys Phe Arg Gln Gln Arg Val Ser Lys Thr Leu Asp 485 490 495 Gln Asn Asn Asn Ser Asn Phe Ala Trp Thr Gly Ala Thr Lys Tyr His 500 505 510 Leu Asn Gly Arg Asn Ser Leu Val Asn Pro Gly Val Ala Met Ala Thr 515 520 525 His Lys Asp Asp Glu Asp Arg Phe Phe Pro Ser Ser Gly Val Leu Ile 530 535 540 Phe Gly Lys Thr Gly Ala Thr Asn Lys Thr Thr Leu Glu Asn Val Leu 545 550 555 560 Met Thr Asn Glu Glu Glu Ile Arg Pro Thr Asn Pro Val Ala Thr Glu 565 570 575 Glu Tyr Gly Ile Val Ser Ser Asn Leu Gln Ala Ala Asn Thr Ala Ala 580 585 590 Gln Thr Gln Val Val Asn Asn Gln Gly Ala Leu Pro Gly Met Val Trp 595 600 605 Gln Asn Arg Asp Val Tyr Leu Gln Gly Pro Ile Trp Ala Lys Ile Pro 610 615 620 His Thr Asp Gly Asn Phe His Pro Ser Pro Leu Met Gly Gly Phe Gly 625 630 635 640 Leu Lys His Pro Pro Pro Gln Ile Leu Ile Lys Asn Thr Pro Val Pro 645 650 655 Ala Asn Pro Pro Glu Val Phe Thr Pro Ala Lys Phe Ala Ser Phe Ile 660 665 670 Thr Gln Tyr Ser Thr Gly Gln Val Ser Val Glu Ile Glu Trp Glu Leu 675 680 685 Gln Lys Glu Asn Ser Lys Arg Trp Asn Pro Glu Ile Gln Tyr Thr Ser 690 695 700 Asn Phe Glu Lys Gln Thr Gly Val Asp Phe Ala Val Asp Ser Gln Gly 705 710 715 720 Val Tyr Ser Glu Pro Arg Pro Ile Gly Thr Arg Tyr Leu Thr Arg Asn 725 730 735 Leu <210> 9 <211> 738 <212> PRT <213> Parvovirus adeno-associated virus <400> 9 Met Ala Ala Asp Gly Tyr Leu Pro Asp Trp Leu Glu Asp Asn Leu Ser 1 5 10 15 Glu Gly Ile Arg Glu Trp Trp Ala Leu Lys Pro Gly Ala Pro Lys Pro 20 25 30 Lys Ala Asn Gln Gln Lys Gln Asp Asp Gly Arg Gly Leu Val Leu Pro 35 40 45 Gly Tyr Lys Tyr Leu Gly Pro Phe Asn Gly Leu Asp Lys Gly Glu Pro 50 55 60 Val Asn Ala Ala Asp Ala Ala Ala Leu Glu His Asp Lys Ala Tyr Asp 65 70 75 80 Gln Gln Leu Gln Ala Gly Asp Asn Pro Tyr Leu Arg Tyr Asn His Ala 85 90 95 Asp Ala Glu Phe Gln Glu Arg Leu Gln Glu Asp Thr Ser Phe Gly Gly 100 105 110 Asn Leu Gly Arg Ala Val Phe Gln Ala Lys Lys Arg Val Leu Glu Pro 115 120 125 Leu Gly Leu Val Glu Glu Gly Ala Lys Thr Ala Pro Gly Lys Lys Arg 130 135 140 Pro Val Glu Pro Ser Pro Gln Arg Ser Pro Asp Ser Ser Thr Gly Ile 145 150 155 160 Gly Lys Lys Gly Gln Gln Pro Ala Arg Lys Arg Leu Asn Phe Gly Gln 165 170 175 Thr Gly Asp Ser Glu Ser Val Pro Asp Pro Gln Pro Leu Gly Glu Pro 180 185 190 Pro Ala Ala Pro Ser Gly Val Gly Pro Asn Thr Met Ala Ala Gly Gly 195 200 205 Gly Ala Pro Met Ala Asp Asn Asn Glu Gly Ala Asp Gly Val Gly Ser 210 215 220 Ser Ser Gly Asn Trp His Cys Asp Ser Thr Trp Leu Gly Asp Arg Val 225 230 235 240 Ile Thr Thr Ser Thr Arg Thr Trp Ala Leu Pro Thr Tyr Asn Asn His 245 250 255 Leu Tyr Lys Gln Ile Ser Asn Gly Thr Ser Gly Gly Ala Thr Asn Asp 260 265 270 Asn Thr Tyr Phe Gly Tyr Ser Thr Pro Trp Gly Tyr Phe Asp Phe Asn 275 280 285 Arg Phe His Cys His Phe Ser Pro Arg Asp Trp Gln Arg Leu Ile Asn 290 295 300 Asn Asn Trp Gly Phe Arg Pro Lys Arg Leu Ser Phe Lys Leu Phe Asn 305 310 315 320 Ile Gln Val Lys Glu Val Thr Gln Asn Glu Gly Thr Lys Thr Ile Ala 325 330 335 Asn Asn Leu Thr Ser Thr Ile Gln Val Phe Thr Asp Ser Glu Tyr Gln 340 345 350 Leu Pro Tyr Val Leu Gly Ser Ala His Gln Gly Cys Leu Pro Pro Phe 355 360 365 Pro Ala Asp Val Phe Met Ile Pro Gln Tyr Gly Tyr Leu Thr Leu Asn 370 375 380 Asn Gly Ser Gln Ala Val Gly Arg Ser Ser Phe Tyr Cys Leu Glu Tyr 385 390 395 400 Phe Pro Ser Gln Met Leu Arg Thr Gly Asn Asn Phe Gln Phe Thr Tyr 405 410 415 Thr Phe Glu Asp Val Pro Phe His Ser Ser Tyr Ala His Ser Gln Ser 420 425 430 Leu Asp Arg Leu Met Asn Pro Leu Ile Asp Gln Tyr Leu Tyr Tyr Leu 435 440 445 Ser Arg Thr Gln Thr Thr Gly Gly Thr Ala Asn Thr Gln Thr Leu Gly 450 455 460 Phe Ser Gln Gly Gly Pro Asn Thr Met Ala Asn Gln Ala Lys Asn Trp 465 470 475 480 Leu Pro Gly Pro Cys Tyr Arg Gln Gln Arg Val Ser Thr Thr Thr Gly 485 490 495 Gln Asn Asn Asn Ser Asn Phe Ala Trp Thr Ala Gly Thr Lys Tyr His 500 505 510 Leu Asn Gly Arg Asn Ser Leu Ala Asn Pro Gly Ile Ala Met Ala Thr 515 520 525 His Lys Asp Asp Glu Glu Arg Phe Phe Pro Ser Asn Gly Ile Leu Ile 530 535 540 Phe Gly Lys Gln Asn Ala Ala Arg Asp Asn Ala Asp Tyr Ser Asp Val 545 550 555 560 Met Leu Thr Ser Glu Glu Glu Ile Lys Thr Thr Asn Pro Val Ala Thr 565 570 575 Glu Glu Tyr Gly Ile Val Ala Asp Asn Leu Gln Gln Gln Asn Thr Ala 580 585 590 Pro Gln Ile Gly Thr Val Asn Ser Gln Gly Ala Leu Pro Gly Met Val 595 600 605 Trp Gln Asn Arg Asp Val Tyr Leu Gln Gly Pro Ile Trp Ala Lys Ile 610 615 620 Pro His Thr Asp Gly Asn Phe His Pro Ser Pro Leu Met Gly Gly Phe 625 630 635 640 Gly Leu Lys His Pro Pro Gln Ile Leu Ile Lys Asn Thr Pro Val 645 650 655 Pro Ala Asp Pro Pro Thr Thr Phe Asn Gln Ser Lys Leu Asn Ser Phe 660 665 670 Ile Thr Gln Tyr Ser Thr Gly Gin Val Ser Val Glu Ile Glu Trp Glu 675 680 685 Leu Gln Lys Glu Asn Ser Lys Arg Trp Asn Pro Glu Ile Gln Tyr Thr 690 695 700 Ser Asn Tyr Tyr Lys Ser Thr Ser Val Asp Phe Ala Val Asn Thr Glu 705 710 715 720 Gly Val Tyr Ser Glu Pro Arg Pro Ile Gly Thr Arg Tyr Leu Thr Arg 725 730 735 Asn Leu <210> 10 <211> 736 <212> PRT <213> Parvovirus adeno-associated virus <400> 10 Met Ala Ala Asp Gly Tyr Leu Pro Asp Trp Leu Glu Asp Asn Leu Ser 1 5 10 15 Glu Gly Ile Arg Glu Trp Trp Ala Leu Lys Pro Gly Ala Pro Gln Pro 20 25 30 Lys Ala Asn Gln Gln His Gln Asp Asn Ala Arg Gly Leu Val Leu Pro 35 40 45 Gly Tyr Lys Tyr Leu Gly Pro Gly Asn Gly Leu Asp Lys Gly Glu Pro 50 55 60 Val Asn Ala Ala Asp Ala Ala Ala Leu Glu His Asp Lys Ala Tyr Asp 65 70 75 80 Gln Gln Leu Lys Ala Gly Asp Asn Pro Tyr Leu Lys Tyr Asn His Ala 85 90 95 Asp Ala Glu Phe Gln Glu Arg Leu Lys Glu Asp Thr Ser Phe Gly Gly 100 105 110 Asn Leu Gly Arg Ala Val Phe Gln Ala Lys Lys Arg Leu Leu Glu Pro 115 120 125 Leu Gly Leu Val Glu Glu Ala Ala Lys Thr Ala Pro Gly Lys Lys Arg 130 135 140 Pro Val Glu Gln Ser Pro Gln Glu Pro Asp Ser Ser Ala Gly Ile Gly 145 150 155 160 Lys Ser Gly Ala Gln Pro Ala Lys Lys Arg Leu Asn Phe Gly Gln Thr 165 170 175 Gly Asp Thr Glu Ser Val Pro Asp Pro Gln Pro Ile Gly Glu Pro Pro 180 185 190 Ala Ala Pro Ser Gly Val Gly Ser Leu Thr Met Ala Ser Gly Gly Gly 195 200 205 Ala Pro Val Ala Asp Asn Asn Glu Gly Ala Asp Gly Val Gly Ser Ser 210 215 220 Ser Gly Asn Trp His Cys Asp Ser Gln Trp Leu Gly Asp Arg Val Ile 225 230 235 240 Thr Thr Ser Thr Arg Thr Trp Ala Leu Pro Thr Tyr Asn Asn His Leu 245 250 255 Tyr Lys Gln Ile Ser Asn Ser Thr Ser Gly Gly Ser Ser Asn Asp Asn 260 265 270 Ala Tyr Phe Gly Tyr Ser Thr Pro Trp Gly Tyr Phe Asp Phe Asn Arg 275 280 285 Phe His Cys His Phe Ser Pro Arg Asp Trp Gln Arg Leu Ile Asn Asn 290 295 300 Asn Trp Gly Phe Arg Pro Lys Arg Leu Asn Phe Lys Leu Phe Asn Ile 305 310 315 320 Gln Val Lys Glu Val Thr Asp Asn Asn Gly Val Lys Thr Ile Ala Asn 325 330 335 Asn Leu Thr Ser Thr Val Gln Val Phe Thr Asp Ser Asp Tyr Gln Leu 340 345 350 Pro Tyr Val Leu Gly Ser Ala His Glu Gly Cys Leu Pro Pro Phe Pro 355 360 365 Ala Asp Val Phe Met Ile Pro Gln Tyr Gly Tyr Leu Thr Leu Asn Asp 370 375 380 Gly Ser Gln Ala Val Gly Arg Ser Ser Phe Tyr Cys Leu Glu Tyr Phe 385 390 395 400 Pro Ser Gln Met Leu Arg Thr Gly Asn Asn Phe Gln Phe Ser Tyr Glu 405 410 415 Phe Glu Asn Val Pro Phe His Ser Ser Tyr Ala His Ser Gln Ser Leu 420 425 430 Asp Arg Leu Met Asn Pro Leu Ile Asp Gln Tyr Leu Tyr Tyr Leu Ser 435 440 445 Lys Thr Ile Asn Gly Ser Gly Gln Asn Gln Gln Thr Leu Lys Phe Ser 450 455 460 Val Ala Gly Pro Ser Asn Met Ala Val Gln Gly Arg Asn Tyr Ile Pro 465 470 475 480 Gly Pro Ser Tyr Arg Gln Gln Arg Val Ser Thr Thr Val Thr Gln Asn 485 490 495 Asn Asn Ser Glu Phe Ala Trp Pro Gly Ala Ser Ser Trp Ala Leu Asn 500 505 510 Gly Arg Asn Ser Leu Met Asn Pro Gly Pro Ala Met Ala Ser His Lys 515 520 525 Glu Gly Glu Asp Arg Phe Phe Pro Leu Ser Gly Ser Leu Ile Phe Gly 530 535 540 Lys Gln Gly Thr Gly Arg Asp Asn Val Asp Ala Asp Lys Val Met Ile 545 550 555 560 Thr Asn Glu Glu Glu Ile Lys Thr Thr Asn Pro Val Ala Thr Glu Ser 565 570 575 Tyr Gly Gln Val Ala Thr Asn His Gln Ser Ala Gln Ala Gln Ala Gln 580 585 590 Thr Gly Trp Val Gln Asn Gln Gly Ile Leu Pro Gly Met Val Trp Gln 595 600 605 Asp Arg Asp Val Tyr Leu Gln Gly Pro Ile Trp Ala Lys Ile Pro His 610 615 620 Thr Asp Gly Asn Phe His Pro Ser Pro Leu Met Gly Gly Phe Gly Met 625 630 635 640 Lys His Pro Pro Pro Gln Ile Leu Ile Lys Asn Thr Pro Val Pro Ala 645 650 655 Asp Pro Pro Thr Ala Phe Asn Lys Asp Lys Leu Asn Ser Phe Ile Thr 660 665 670 Gln Tyr Ser Thr Gly Gln Val Ser Val Glu Ile Glu Trp Glu Leu Gln 675 680 685 Lys Glu Asn Ser Lys Arg Trp Asn Pro Glu Ile Gln Tyr Thr Ser Asn 690 695 700 Tyr Tyr Lys Ser Asn Asn Val Glu Phe Ala Val Asn Thr Glu Gly Val 705 710 715 720 Tyr Ser Glu Pro Arg Pro Ile Gly Thr Arg Tyr Leu Thr Arg Asn Leu 725 730 735 <210> 11 <211> 738 <212> PRT <213> Parvovirus adeno-associated virus <400> 11 Met Ala Ala Asp Gly Tyr Leu Pro Asp Trp Leu Glu Asp Asn Leu Ser 1 5 10 15 Glu Gly Ile Arg Glu Trp Trp Asp Leu Lys Pro Gly Ala Pro Lys Pro 20 25 30 Lys Ala Asn Gln Gln Lys Gln Asp Asp Gly Arg Gly Leu Val Leu Pro 35 40 45 Gly Tyr Lys Tyr Leu Gly Pro Phe Asn Gly Leu Asp Lys Gly Glu Pro 50 55 60 Val Asn Ala Ala Asp Ala Ala Ala Leu Glu His Asp Lys Ala Tyr Asp 65 70 75 80 Gln Gln Leu Lys Ala Gly Asp Asn Pro Tyr Leu Arg Tyr Asn His Ala 85 90 95 Asp Ala Glu Phe Gln Glu Arg Leu Gln Glu Asp Thr Ser Phe Gly Gly 100 105 110 Asn Leu Gly Arg Ala Val Phe Gln Ala Lys Lys Arg Val Leu Glu Pro 115 120 125 Leu Gly Leu Val Glu Glu Gly Ala Lys Thr Ala Pro Gly Lys Lys Arg 130 135 140 Pro Val Glu Pro Ser Pro Gln Arg Ser Pro Asp Ser Ser Thr Gly Ile 145 150 155 160 Gly Lys Lys Gly Gln Gln Pro Ala Lys Lys Arg Leu Asn Phe Gly Gln 165 170 175 Thr Gly Asp Ser Glu Ser Val Pro Asp Pro Gln Pro Ile Gly Glu Pro 180 185 190 Pro Ala Gly Pro Ser Gly Leu Gly Ser Gly Thr Met Ala Ala Gly Gly 195 200 205 Gly Ala Pro Met Ala Asp Asn Asn Glu Gly Ala Asp Gly Val Gly Ser 210 215 220 Ser Ser Gly Asn Trp His Cys Asp Ser Thr Trp Leu Gly Asp Arg Val 225 230 235 240 Ile Thr Thr Ser Thr Arg Thr Trp Ala Leu Pro Thr Tyr Asn Asn His 245 250 255 Leu Tyr Lys Gln Ile Ser Asn Gly Thr Ser Gly Gly Ser Thr Asn Asp 260 265 270 Asn Thr Tyr Phe Gly Tyr Ser Thr Pro Trp Gly Tyr Phe Asp Phe Asn 275 280 285 Arg Phe His Cys His Phe Ser Pro Arg Asp Trp Gln Arg Leu Ile Asn 290 295 300 Asn Asn Trp Gly Phe Arg Pro Lys Arg Leu Asn Phe Lys Leu Phe Asn 305 310 315 320 Ile Gln Val Lys Glu Val Thr Gln Asn Glu Gly Thr Lys Thr Ile Ala 325 330 335 Asn Asn Leu Thr Ser Thr Ile Gln Val Phe Thr Asp Ser Glu Tyr Gln 340 345 350 Leu Pro Tyr Val Leu Gly Ser Ala His Gln Gly Cys Leu Pro Pro Phe 355 360 365 Pro Ala Asp Val Phe Met Ile Pro Gln Tyr Gly Tyr Leu Thr Leu Asn 370 375 380 Asn Gly Ser Gln Ala Val Gly Arg Ser Ser Phe Tyr Cys Leu Glu Tyr 385 390 395 400 Phe Pro Ser Gln Met Leu Arg Thr Gly Asn Asn Phe Glu Phe Ser Tyr 405 410 415 Gln Phe Glu Asp Val Pro Phe His Ser Ser Tyr Ala His Ser Gln Ser 420 425 430 Leu Asp Arg Leu Met Asn Pro Leu Ile Asp Gln Tyr Leu Tyr Tyr Leu 435 440 445 Ser Arg Thr Gln Ser Thr Gly Gly Thr Ala Gly Thr Gln Gln Leu Leu 450 455 460 Phe Ser Gln Ala Gly Pro Asn Asn Met Ser Ala Gln Ala Lys Asn Trp 465 470 475 480 Leu Pro Gly Pro Cys Tyr Arg Gln Gln Arg Val Ser Thr Thr Leu Ser 485 490 495 Gln Asn Asn Asn Ser Asn Phe Ala Trp Thr Gly Ala Thr Lys Tyr His 500 505 510 Leu Asn Gly Arg Asp Ser Leu Val Asn Pro Gly Val Ala Met Ala Thr 515 520 525 His Lys Asp Asp Glu Glu Arg Phe Phe Pro Ser Ser Gly Val Leu Met 530 535 540 Phe Gly Lys Gln Gly Ala Gly Lys Asp Asn Val Asp Tyr Ser Ser Val 545 550 555 560 Met Leu Thr Ser Glu Glu Glu Ile Lys Thr Thr Asn Pro Val Ala Thr 565 570 575 Glu Gln Tyr Gly Val Val Ala Asp Asn Leu Gln Gln Gln Asn Ala Ala 580 585 590 Pro Ile Val Gly Ala Val Asn Ser Gln Gly Ala Leu Pro Gly Met Val 595 600 605 Trp Gln Asn Arg Asp Val Tyr Leu Gln Gly Pro Ile Trp Ala Lys Ile 610 615 620 Pro His Thr Asp Gly Asn Phe His Pro Ser Pro Leu Met Gly Gly Phe 625 630 635 640 Gly Leu Lys His Pro Pro Gln Ile Leu Ile Lys Asn Thr Pro Val 645 650 655 Pro Ala Asp Pro Pro Thr Thr Phe Ser Gln Ala Lys Leu Ala Ser Phe 660 665 670 Ile Thr Gln Tyr Ser Thr Gly Gin Val Ser Val Glu Ile Glu Trp Glu 675 680 685 Leu Gln Lys Glu Asn Ser Lys Arg Trp Asn Pro Glu Ile Gln Tyr Thr 690 695 700 Ser Asn Tyr Tyr Lys Ser Thr Asn Val Asp Phe Ala Val Asn Thr Asp 705 710 715 720 Gly Thr Tyr Ser Glu Pro Arg Pro Ile Gly Thr Arg Tyr Leu Thr Arg 725 730 735 Asn Leu <210> 12 <211> 733 <212> PRT <213> Parvovirus adeno-associated virus <400> 12 Met Ala Ala Asp Gly Tyr Leu Pro Asp Trp Leu Glu Asp Asn Leu Ser 1 5 10 15 Glu Gly Ile Arg Glu Trp Trp Asp Leu Lys Pro Gly Ala Pro Lys Pro 20 25 30 Lys Ala Asn Gln Gln Lys Gln Asp Asp Gly Arg Gly Leu Val Leu Pro 35 40 45 Gly Tyr Lys Tyr Leu Gly Pro Phe Asn Gly Leu Asp Lys Gly Glu Pro 50 55 60 Val Asn Ala Ala Asp Ala Ala Ala Leu Glu His Asp Lys Ala Tyr Asp 65 70 75 80 Gln Gln Leu Lys Ala Gly Asp Asn Pro Tyr Leu Arg Tyr Asn His Ala 85 90 95 Asp Ala Glu Phe Gln Glu Arg Leu Gln Glu Asp Thr Ser Phe Gly Gly 100 105 110 Asn Leu Gly Arg Ala Val Phe Gln Ala Lys Lys Arg Val Leu Glu Pro 115 120 125 Leu Gly Leu Val Glu Glu Gly Ala Lys Thr Ala Pro Gly Lys Lys Arg 130 135 140 Pro Leu Glu Ser Pro Gln Glu Pro Asp Ser Ser Ser Gly Ile Gly Lys 145 150 155 160 Lys Gly Lys Gln Pro Ala Arg Lys Arg Leu Asn Phe Glu Glu Asp Thr 165 170 175 Gly Ala Gly Asp Gly Pro Pro Glu Gly Ser Asp Thr Ser Ala Met Ser 180 185 190 Ser Asp Ile Glu Met Arg Ala Ala Pro Gly Gly Asn Ala Val Asp Ala 195 200 205 Gly Gln Gly Ser Asp Gly Val Gly Asn Ala Ser Gly Asp Trp His Cys 210 215 220 Asp Ser Thr Trp Ser Glu Gly Lys Val Thr Thr Thr Ser Thr Arg Thr 225 230 235 240 Trp Val Leu Pro Thr Tyr Asn Asn His Leu Tyr Leu Arg Leu Gly Thr 245 250 255 Thr Ser Ser Ser Asn Thr Tyr Asn Gly Phe Ser Thr Pro Trp Gly Tyr 260 265 270 Phe Asp Phe Asn Arg Phe His Cys His Phe Ser Pro Arg Asp Trp Gln 275 280 285 Arg Leu Ile Asn Asn Asn Trp Gly Leu Arg Pro Lys Ala Met Arg Val 290 295 300 Lys Ile Phe Asn Ile Gln Val Lys Glu Val Thr Thr Ser Asn Gly Glu 305 310 315 320 Thr Thr Val Ala Asn Asn Leu Thr Ser Thr Val Gln Ile Phe Ala Asp 325 330 335 Ser Ser Tyr Glu Leu Pro Tyr Val Met Asp Ala Gly Gln Glu Gly Ser 340 345 350 Leu Pro Pro Phe Pro Asn Asp Val Phe Met Val Pro Gln Tyr Gly Tyr 355 360 365 Cys Gly Ile Val Thr Gly Glu Asn Gln Asn Gln Thr Asp Arg Asn Ala 370 375 380 Phe Tyr Cys Leu Glu Tyr Phe Pro Ser Gln Met Leu Arg Thr Gly Asn 385 390 395 400 Asn Phe Glu Met Ala Tyr Asn Phe Glu Lys Val Pro Phe His Ser Met 405 410 415 Tyr Ala His Ser Gln Ser Leu Asp Arg Leu Met Asn Pro Leu Leu Asp 420 425 430 Gln Tyr Leu Trp His Leu Gln Ser Thr Thr Ser Gly Glu Thr Leu Asn 435 440 445 Gln Gly Asn Ala Ala Thr Thr Phe Gly Lys Ile Arg Ser Gly Asp Phe 450 455 460 Ala Phe Tyr Arg Lys Asn Trp Leu Pro Gly Pro Cys Val Lys Gln Gln 465 470 475 480 Arg Phe Ser Lys Thr Ala Ser Gln Asn Tyr Lys Ile Pro Ala Ser Gly 485 490 495 Gly Asn Ala Leu Leu Lys Tyr Asp Thr His Tyr Thr Leu Asn Asn Arg 500 505 510 Trp Ser Asn Ile Ala Pro Gly Pro Pro Met Ala Thr Ala Gly Pro Ser 515 520 525 Asp Gly Asp Phe Ser Asn Ala Gln Leu Ile Phe Pro Gly Pro Ser Val 530 535 540 Thr Gly Asn Thr Thr Thr Ser Ala Asn Asn Leu Leu Phe Thr Ser Glu 545 550 555 560 Glu Glu Ile Ala Ala Thr Asn Pro Arg Asp Thr Asp Met Phe Gly Gln 565 570 575 Ile Ala Asp Asn Asn Gln Asn Ala Thr Thr Ala Pro Ile Thr Gly Asn 580 585 590 Val Thr Ala Met Gly Val Leu Pro Gly Met Val Trp Gln Asn Arg Asp 595 600 605 Ile Tyr Tyr Gln Gly Pro Ile Trp Ala Lys Ile Pro His Ala Asp Gly 610 615 620 His Phe His Pro Ser Pro Leu Ile Gly Gly Phe Gly Leu Lys His Pro 625 630 635 640 Pro Pro Gln Ile Phe Ile Lys Asn Thr Pro Val Pro Ala Asn Pro Ala 645 650 655 Thr Thr Phe Thr Ala Ala Arg Val Asp Ser Phe Ile Thr Gln Tyr Ser 660 665 670 Thr Gly Gln Val Ala Val Gln Ile Glu Trp Glu Ile Glu Lys Glu Arg 675 680 685 Ser Lys Arg Trp Asn Pro Glu Val Gln Phe Thr Ser Asn Tyr Gly Asn 690 695 700 Gln Ser Ser Met Leu Trp Ala Pro Asp Thr Thr Gly Lys Tyr Thr Glu 705 710 715 720 Pro Arg Val Ile Gly Ser Arg Tyr Leu Thr Asn His Leu 725 730 <210> 13 <211> 341 <212> PRT <213> Streptococcus pyogenes <400> 13 Met Arg Lys Arg Cys Tyr Ser Thr Ser Ala Ala Val Leu Ala Ala Val 1 5 10 15 Thr Leu Phe Ala Leu Ser Val Asp Arg Gly Val Ile Ala Asp Ser Phe 20 25 30 Ser Ala Asn Gln Glu Ile Arg Tyr Ser Glu Val Thr Pro Tyr His Val 35 40 45 Thr Ser Val Trp Thr Lys Gly Val Thr Pro Pro Ala Lys Phe Thr Gln 50 55 60 Gly Glu Asp Val Phe His Ala Pro Tyr Val Ala Asn Gln Gly Trp Tyr 65 70 75 80 Asp Ile Thr Lys Thr Phe Asn Gly Lys Asp Asp Leu Leu Cys Gly Ala 85 90 95 Ala Thr Ala Gly Asn Met Leu His Trp Trp Phe Asp Gln Asn Lys Glu 100 105 110 Lys Ile Glu Ala Tyr Leu Lys Lys His Pro Asp Lys Gln Lys Ile Met 115 120 125 Phe Gly Asp Gln Glu Leu Leu Asp Val Arg Lys Val Ile Asn Thr Lys 130 135 140 Gly Asp Gln Thr Asn Ser Glu Leu Phe Asn Tyr Phe Arg Asp Lys Ala 145 150 155 160 Phe Pro Gly Leu Ser Ala Arg Arg Ile Gly Val Met Pro Asp Leu Val 165 170 175 Leu Asp Met Phe Ile Asn Gly Tyr Tyr Leu Asn Val Tyr Lys Thr Gln 180 185 190 Thr Thr Asp Val Asn Arg Thr Tyr Gln Glu Lys Asp Arg Arg Gly Gly 195 200 205 Ile Phe Asp Ala Val Phe Thr Arg Gly Asp Gln Ser Lys Leu Leu Thr 210 215 220 Ser Arg His Asp Phe Lys Glu Lys Asn Leu Lys Glu Ile Ser Asp Leu 225 230 235 240 Ile Lys Lys Glu Leu Thr Glu Gly Lys Ala Leu Gly Leu Ser His Thr 245 250 255 Tyr Ala Asn Val Arg Ile Asn His Val Ile Asn Leu Trp Gly Ala Asp 260 265 270 Phe Asp Ser Asn Gly Asn Leu Lys Ala Ile Tyr Val Thr Asp Ser Asp 275 280 285 Ser Asn Ala Ser Ile Gly Met Lys Lys Tyr Phe Val Gly Val Asn Ser 290 295 300 Ser Gly Lys Val Ala Ile Ser Ala Lys Glu Ile Lys Glu Asp Asn Ile 305 310 315 320 Gly Ala Gln Val Leu Gly Leu Phe Thr Leu Ser Thr Gly Gln Asp Ser 325 330 335 Trp Asn Gln Thr Asn 340 <210> 14 <211> 341 <212> PRT <213> Streptococcus pyogenes <400> 14 Met Arg Lys Arg Cys Tyr Ser Thr Ser Ala Ala Val Leu Ala Ala Val 1 5 10 15 Thr Leu Phe Ala Leu Ser Val Asp Arg Gly Val Ile Ala Asp Ser Phe 20 25 30 Ser Ala Asn Gln Glu Ile Arg Tyr Ser Glu Val Thr Pro Tyr His Val 35 40 45 Thr Ser Val Trp Thr Lys Gly Val Thr Pro Pro Ala Lys Phe Thr Gln 50 55 60 Gly Glu Asp Val Phe His Ala Pro Tyr Val Ala Asn Gln Gly Trp Tyr 65 70 75 80 Asp Ile Thr Lys Thr Phe Asn Gly Lys Asp Asp Leu Leu Cys Gly Ala 85 90 95 Ala Thr Ala Gly Asn Met Leu His Trp Trp Phe Asp Gln Asn Lys Glu 100 105 110 Lys Ile Glu Ala Tyr Leu Lys Lys His Pro Asp Lys Gln Lys Ile Met 115 120 125 Phe Gly Asp Gln Glu Leu Leu Asp Val Arg Lys Val Ile Asn Thr Lys 130 135 140 Gly Asp Gln Thr Asn Ser Glu Leu Phe Asn Tyr Phe Arg Asp Lys Ala 145 150 155 160 Phe Pro Gly Leu Ser Ala Arg Arg Ile Gly Val Met Pro Asp Leu Val 165 170 175 Leu Asp Met Phe Ile Asn Gly Tyr Tyr Leu Asn Val Tyr Lys Thr Gln 180 185 190 Thr Thr Asp Val Asn Arg Thr Tyr Gln Glu Lys Asp Arg Arg Gly Gly 195 200 205 Ile Phe Asp Ala Val Phe Thr Arg Gly Asp Gln Ser Lys Leu Leu Thr 210 215 220 Ser Arg His Asp Phe Lys Glu Lys Asn Leu Lys Glu Ile Ser Asp Leu 225 230 235 240 Ile Lys Lys Glu Leu Thr Glu Gly Lys Ala Leu Gly Leu Ser His Thr 245 250 255 Tyr Ala Asn Val Arg Ile Asn His Val Ile Asn Leu Trp Gly Ala Asp 260 265 270 Phe Asp Ser Asn Gly Asn Leu Lys Ala Ile Tyr Val Thr Asp Ser Asp 275 280 285 Ser Asn Ala Ser Ile Gly Met Lys Lys Tyr Phe Val Gly Val Asn Ser 290 295 300 Ser Gly Lys Val Ala Ile Ser Ala Lys Glu Ile Lys Glu Asp Asn Ile 305 310 315 320 Gly Ala Gln Val Leu Gly Leu Phe Thr Leu Ser Thr Gly Gln Asp Ser 325 330 335 Trp Asn Gln Thr Asn 340 <210> 15 <211> 339 <212> PRT <213> Artificial Sequence <220> <223> IgG degrading enzyme amino acid sequence <400> 15 Met Arg Lys Arg Cys Tyr Ser Thr Ser Ala Ala Val Leu Ala Ala Val 1 5 10 15 Thr Leu Phe Val Leu Ser Val Asp Arg Gly Val Ile Ala Asp Ser Phe 20 25 30 Ser Ala Asn Gln Glu Ile Arg Tyr Ser Glu Val Thr Pro Tyr His Val 35 40 45 Thr Ser Val Trp Thr Lys Gly Val Thr Pro Pro Ala Asn Phe Thr Gln 50 55 60 Gly Glu Asp Val Phe His Ala Pro Tyr Val Ala Asn Gln Gly Trp Tyr 65 70 75 80 Asp Ile Thr Lys Thr Phe Asn Gly Lys Asp Asp Leu Leu Cys Gly Ala 85 90 95 Ala Thr Ala Gly Asn Met Leu His Trp Trp Phe Asp Gln Asn Lys Asp 100 105 110 Gln Ile Lys Arg Tyr Leu Glu Glu His Pro Glu Lys Gln Lys Ile Asn 115 120 125 Phe Asn Gly Glu Gln Met Phe Asp Val Lys Glu Ala Ile Asp Thr Lys 130 135 140 Asn His Gln Leu Asp Ser Lys Leu Phe Glu Tyr Phe Lys Glu Lys Ala 145 150 155 160 Phe Pro Tyr Leu Ser Thr Lys His Leu Gly Val Phe Pro Asp His Val 165 170 175 Ile Asp Met Phe Ile Asn Gly Tyr Arg Leu Ser Leu Thr Asn His Gly 180 185 190 Pro Thr Pro Val Lys Glu Gly Ser Lys Asp Pro Arg Gly Gly Ile Phe 195 200 205 Asp Ala Val Phe Thr Arg Gly Asp Gln Ser Lys Leu Leu Thr Ser Arg 210 215 220 His Asp Phe Lys Glu Lys Asn Leu Lys Glu Ile Ser Asp Leu Ile Lys 225 230 235 240 Lys Glu Leu Thr Glu Gly Lys Ala Leu Gly Leu Ser His Thr Tyr Ala 245 250 255 Asn Val Arg Ile Asn His Val Ile Asn Leu Trp Gly Ala Asp Phe Asp 260 265 270 Ser Asn Gly Asn Leu Lys Ala Ile Tyr Val Thr Asp Ser Asp Ser Asn 275 280 285 Ala Ser Ile Gly Met Lys Lys Tyr Phe Val Gly Val Asn Ser Ala Gly 290 295 300 Lys Val Ala Ile Ser Ala Lys Glu Ile Lys Glu Asp Asn Ile Gly Ala 305 310 315 320 Gln Val Leu Gly Leu Phe Thr Leu Ser Thr Gly Gln Asp Ser Trp Asn 325 330 335 Gln Thr Asn <210> 16 <211> 310 <212> PRT <213> Artificial Sequence <220> <223> IgG degrading enzyme amino acid sequence <400> 16 Asp Ser Phe Ser Ala Asn Gln Glu Ile Arg Tyr Ser Glu Val Thr Pro 1 5 10 15 Tyr His Val Thr Ser Val Trp Thr Lys Gly Val Thr Pro Pro Ala Asn 20 25 30 Phe Thr Gln Gly Glu Asp Val Phe His Ala Pro Tyr Val Ala Asn Gln 35 40 45 Gly Trp Tyr Asp Ile Thr Lys Thr Phe Asn Gly Lys Asp Asp Leu Leu 50 55 60 Cys Gly Ala Ala Thr Ala Gly Asn Met Leu His Trp Trp Phe Asp Gln 65 70 75 80 Asn Lys Asp Gln Ile Lys Arg Tyr Leu Glu Glu His Pro Glu Lys Gln 85 90 95 Lys Ile Asn Phe Asn Gly Glu Gln Met Phe Asp Val Lys Glu Ala Ile 100 105 110 Asp Thr Lys Asn His Gln Leu Asp Ser Lys Leu Phe Glu Tyr Phe Lys 115 120 125 Glu Lys Ala Phe Pro Tyr Leu Ser Thr Lys His Leu Gly Val Phe Pro 130 135 140 Asp His Val Ile Asp Met Phe Ile Asn Gly Tyr Arg Leu Ser Leu Thr 145 150 155 160 Asn His Gly Pro Thr Pro Val Lys Glu Gly Ser Lys Asp Pro Arg Gly 165 170 175 Gly Ile Phe Asp Ala Val Phe Thr Arg Gly Asp Gln Ser Lys Leu Leu 180 185 190 Thr Ser Arg His Asp Phe Lys Glu Lys Asn Leu Lys Glu Ile Ser Asp 195 200 205 Leu Ile Lys Lys Glu Leu Thr Glu Gly Lys Ala Leu Gly Leu Ser His 210 215 220 Thr Tyr Ala Asn Val Arg Ile Asn His Val Ile Asn Leu Trp Gly Ala 225 230 235 240 Asp Phe Asp Ser Asn Gly Asn Leu Lys Ala Ile Tyr Val Thr Asp Ser 245 250 255 Asp Ser Asn Ala Ser Ile Gly Met Lys Lys Tyr Phe Val Gly Val Asn 260 265 270 Ser Ala Gly Lys Val Ala Ile Ser Ala Lys Glu Ile Lys Glu Asp Asn 275 280 285 Ile Gly Ala Gln Val Leu Gly Leu Phe Thr Leu Ser Thr Gly Gln Asp 290 295 300 Ser Trp Asn Gln Thr Asn 305 310 <210> 17 <211> 310 <212> PRT <213> Artificial Sequence <220> <223> IgG degrading enzyme amino acid sequence <400> 17 Asp Ser Phe Ser Ala Asn Gln Glu Ile Arg Tyr Ser Glu Val Thr Pro 1 5 10 15 Tyr His Val Thr Ser Val Trp Thr Lys Gly Val Thr Pro Pro Ala Asn 20 25 30 Phe Thr Gln Gly Glu Asp Val Phe His Ala Pro Tyr Val Ala Asn Gln 35 40 45 Gly Trp Tyr Asp Ile Thr Lys Thr Phe Asn Gly Lys Asp Asp Leu Leu 50 55 60 Cys Gly Ala Ala Thr Ala Gly Asn Met Leu His Trp Trp Phe Asp Gln 65 70 75 80 Asn Lys Asp Gln Ile Lys Arg Tyr Leu Glu Glu His Pro Glu Lys Gln 85 90 95 Lys Ile Asn Phe Arg Gly Glu Gln Met Phe Asp Val Lys Glu Ala Ile 100 105 110 Asp Thr Lys Asn His Gln Leu Asp Ser Lys Leu Phe Glu Tyr Phe Lys 115 120 125 Glu Lys Ala Phe Pro Tyr Leu Ser Thr Lys His Leu Gly Val Phe Pro 130 135 140 Asp His Val Ile Asp Met Phe Ile Asn Gly Tyr Arg Leu Ser Leu Thr 145 150 155 160 Asn His Gly Pro Thr Pro Val Lys Glu Gly Ser Lys Asp Pro Arg Gly 165 170 175 Gly Ile Phe Asp Ala Val Phe Thr Arg Gly Asp Gln Ser Lys Leu Leu 180 185 190 Thr Ser Arg His Asp Phe Lys Glu Lys Asn Leu Lys Glu Ile Ser Asp 195 200 205 Leu Ile Lys Lys Glu Leu Thr Glu Gly Lys Ala Leu Gly Leu Ser His 210 215 220 Thr Tyr Ala Asn Val Arg Ile Asn His Val Ile Asn Leu Trp Gly Ala 225 230 235 240 Asp Phe Asp Ser Asn Gly Asn Leu Lys Ala Ile Tyr Val Thr Asp Ser 245 250 255 Asp Ser Asn Ala Ser Ile Gly Met Lys Lys Tyr Phe Val Gly Val Asn 260 265 270 Ser Ala Gly Lys Val Ala Ile Ser Ala Lys Glu Ile Lys Glu Asp Asn 275 280 285 Ile Gly Ala Gln Val Leu Gly Leu Phe Thr Leu Ser Thr Gly Gln Asp 290 295 300 Ser Trp Asn Gln Thr Asn 305 310 <210> 18 <211> 310 <212> PRT <213> Artificial Sequence <220> <223> IgG degrading enzyme amino acid sequence <400> 18 Asp Ser Phe Ser Ala Asn Gln Glu Ile Arg Tyr Ser Glu Val Thr Pro 1 5 10 15 Tyr His Val Thr Ser Val Trp Thr Lys Gly Val Thr Pro Pro Ala Asn 20 25 30 Phe Thr Gln Gly Glu Asp Val Phe His Ala Pro Tyr Val Ala Asn Gln 35 40 45 Gly Trp Tyr Asp Ile Thr Lys Thr Phe Asn Gly Lys Asp Asp Leu Leu 50 55 60 Cys Gly Ala Ala Thr Ala Gly Asn Met Leu His Trp Trp Phe Asp Gln 65 70 75 80 Asn Lys Asp Gln Ile Lys Arg Tyr Leu Glu Glu His Pro Glu Lys Gln 85 90 95 Lys Ile Asn Phe Lys Gly Glu Gln Met Phe Asp Val Lys Glu Ala Ile 100 105 110 Asp Thr Lys Asn His Gln Leu Asp Ser Lys Leu Phe Glu Tyr Phe Lys 115 120 125 Glu Lys Ala Phe Pro Tyr Leu Ser Thr Lys His Leu Gly Val Phe Pro 130 135 140 Asp His Val Ile Asp Met Phe Ile Asn Gly Tyr Arg Leu Ser Leu Thr 145 150 155 160 Asn His Gly Pro Thr Pro Val Lys Arg Gly Ser Lys Asp Pro Arg Gly 165 170 175 Gly Ile Phe Asp Ala Val Phe Thr Arg Gly Asn Gln Ser Lys Leu Leu 180 185 190 Thr Ser Arg His Asp Phe Lys Glu Lys Asn Leu Lys Glu Ile Ser Asp 195 200 205 Leu Ile Lys Lys Glu Leu Thr Glu Gly Lys Ala Leu Gly Leu Ser His 210 215 220 Thr Tyr Ala Asn Val Arg Ile Asn His Val Ile Asn Leu Trp Gly Ala 225 230 235 240 Asp Phe Asp Ser Asn Gly Asn Leu Lys Ala Ile Tyr Val Thr Asp Ser 245 250 255 Asp Ser Asn Ala Ser Ile Gly Met Lys Lys Tyr Phe Val Gly Val Asn 260 265 270 Ser Ala Gly Lys Val Ala Ile Ser Ala Lys Glu Ile Lys Glu Asp Asn 275 280 285 Ile Gly Ala Gln Val Leu Gly Leu Phe Thr Leu Ser Thr Gly Gln Asp 290 295 300 Ser Trp Asn Gln Thr Asn 305 310 <210> 19 <211> 310 <212> PRT <213> Artificial Sequence <220> <223> IgG degrading enzyme amino acid sequence <400> 19 Asp Ser Phe Ser Ala Asn Gln Glu Ile Arg Tyr Ser Glu Val Thr Pro 1 5 10 15 Tyr His Val Thr Ser Val Trp Thr Lys Gly Val Thr Pro Pro Ala Asn 20 25 30 Phe Thr Gln Gly Glu Asp Val Phe His Ala Pro Tyr Val Ala Asn Gln 35 40 45 Gly Trp Tyr Asp Ile Thr Lys Thr Phe Asn Gly Lys Asp Asp Leu Leu 50 55 60 Cys Gly Ala Ala Thr Ala Gly Asn Met Leu His Trp Trp Phe Asp Gln 65 70 75 80 Asn Lys Asp Gln Ile Lys Arg Tyr Leu Glu Glu His Pro Glu Lys Gln 85 90 95 Lys Ile Asn Phe Arg Gly Glu Gln Met Phe Asp Val Lys Glu Ala Ile 100 105 110 Asp Thr Lys Asn His Gln Leu Asp Ser Lys Leu Phe Glu Tyr Phe Lys 115 120 125 Glu Lys Ala Phe Pro Tyr Leu Ser Thr Lys His Leu Gly Val Phe Pro 130 135 140 Asp His Val Ile Asp Met Phe Ile Asn Gly Tyr Arg Leu Ser Leu Thr 145 150 155 160 Asn His Gly Pro Thr Pro Val Lys Lys Gly Ser Lys Asp Pro Arg Gly 165 170 175 Gly Ile Phe Asp Ala Val Phe Thr Arg Gly Asn Gln Ser Lys Leu Leu 180 185 190 Thr Ser Arg His Asp Phe Lys Glu Lys Asn Leu Lys Glu Ile Ser Asp 195 200 205 Leu Ile Lys Lys Glu Leu Thr Glu Gly Lys Ala Leu Gly Leu Ser His 210 215 220 Thr Tyr Ala Asn Val Arg Ile Asn His Val Ile Asn Leu Trp Gly Ala 225 230 235 240 Asp Phe Asp Ser Asn Gly Asn Leu Lys Ala Ile Tyr Val Thr Asp Ser 245 250 255 Asp Ser Asn Ala Ser Ile Gly Met Lys Lys Tyr Phe Val Gly Val Asn 260 265 270 Lys Ala Gly Lys Val Ala Ile Ser Ala Lys Glu Ile Lys Glu Asp Asn 275 280 285 Ile Gly Ala Gln Val Leu Gly Leu Phe Thr Leu Ser Thr Gly Gln Asp 290 295 300 Ser Trp Asn Gln Thr Asn 305 310 <210> 20 <211> 310 <212> PRT <213> Artificial Sequence <220> <223> IgG degrading enzyme amino acid sequence <400> 20 Asp Ser Phe Ser Ala Asn Gln Glu Ile Arg Tyr Ser Glu Val Thr Pro 1 5 10 15 Tyr His Val Thr Ser Val Trp Thr Lys Gly Val Thr Pro Pro Ala Asn 20 25 30 Phe Thr Gln Gly Glu Asp Val Phe His Ala Pro Tyr Val Ala Asn Gln 35 40 45 Gly Trp Tyr Asp Ile Thr Lys Thr Phe Asn Gly Lys Asp Asp Leu Leu 50 55 60 Cys Gly Ala Ala Thr Ala Gly Asn Met Leu His Trp Trp Phe Asp Gln 65 70 75 80 Asn Lys Asp Gln Ile Lys Arg Tyr Leu Arg Glu His Pro Glu Lys Gln 85 90 95 Lys Ile Asn Phe Asn Gly Glu Gln Met Phe Asp Val Lys Glu Ala Ile 100 105 110 Asp Thr Lys Asn His Gln Leu Asp Ser Lys Leu Phe Glu Tyr Phe Lys 115 120 125 Glu Lys Ala Phe Pro Tyr Leu Ser Thr Lys His Leu Gly Val Phe Pro 130 135 140 Asp His Val Ile Asp Met Phe Ile Asn Gly Tyr Arg Leu Ser Leu Thr 145 150 155 160 Asn His Gly Pro Thr Pro Val Lys Glu Gly Ser Lys Asp Pro Arg Gly 165 170 175 Gly Ile Phe Asp Ala Val Phe Thr Arg Gly Asn Gln Ser Lys Leu Leu 180 185 190 Thr Ser Arg His Asp Phe Lys Glu Lys Asn Leu Lys Glu Ile Ser Asp 195 200 205 Leu Ile Lys Lys Glu Leu Asp Glu Gly Lys Ala Leu Gly Leu Ser His 210 215 220 Thr Tyr Ala Asn Val Arg Ile Asn His Val Ile Asn Leu Trp Gly Ala 225 230 235 240 Asp Phe Asp Ser Asn Gly Asn Leu Lys Ala Ile Tyr Val Thr Asp Ser 245 250 255 Asp Ser Asn Ala Ser Ile Gly Met Lys Lys Tyr Phe Val Gly Val Asn 260 265 270 Ser Ala Gly Lys Val Ala Ile Ser Ala Lys Glu Ile Lys Glu Asp Asn 275 280 285 Ile Gly Ala Gln Val Leu Gly Leu Phe Thr Leu Ser Thr Gly Gln Asp 290 295 300 Ser Trp Asn Gln Thr Asn 305 310 <210> 21 <211> 310 <212> PRT <213> Artificial Sequence <220> <223> IgG degrading enzyme amino acid sequence <400> 21 Asp Ser Phe Ser Ala Asn Gln Glu Ile Arg Tyr Ser Glu Val Thr Pro 1 5 10 15 Tyr His Val Thr Ser Val Trp Thr Lys Gly Val Thr Pro Pro Ala Asn 20 25 30 Phe Thr Gln Gly Glu Asp Val Phe His Ala Pro Tyr Val Ala Asn Gln 35 40 45 Gly Trp Tyr Asp Ile Thr Lys Thr Phe Asn Gly Lys Asp Asp Leu Leu 50 55 60 Cys Gly Ala Ala Thr Ala Gly Asn Met Leu His Trp Trp Phe Asp Gln 65 70 75 80 Asn Lys Asp Gln Ile Lys Arg Tyr Leu Lys Glu His Pro Glu Lys Gln 85 90 95 Lys Ile Asn Phe Asn Gly Glu Gln Met Phe Asp Val Lys Glu Ala Ile 100 105 110 Arg Thr Lys Asn His Gln Leu Asp Ser Lys Leu Phe Glu Tyr Phe Lys 115 120 125 Glu Lys Ala Phe Pro Tyr Leu Ser Thr Lys His Leu Gly Val Phe Pro 130 135 140 Asp His Val Ile Asp Met Phe Ile Asn Gly Tyr Arg Leu Ser Leu Thr 145 150 155 160 Asn His Gly Pro Thr Pro Val Lys Glu Gly Ser Lys Asp Pro Arg Gly 165 170 175 Gly Ile Phe Asp Ala Val Phe Thr Arg Gly Asn Gln Ser Lys Leu Leu 180 185 190 Thr Ser Arg His Asp Phe Lys Glu Lys Asn Leu Lys Glu Ile Ser Asp 195 200 205 Leu Ile Lys Lys Glu Leu Glu Glu Gly Lys Ala Leu Gly Leu Ser His 210 215 220 Thr Tyr Ala Asn Val Arg Ile Asn His Val Ile Asn Leu Trp Gly Ala 225 230 235 240 Asp Phe Asp Ser Asn Gly Asn Leu Lys Ala Ile Tyr Val Thr Asp Ser 245 250 255 Asp Ser Asn Ala Ser Ile Gly Met Lys Lys Tyr Phe Val Gly Val Asn 260 265 270 Lys Ala Gly Lys Val Ala Ile Ser Ala Lys Glu Ile Lys Glu Asp Asn 275 280 285 Ile Gly Ala Gln Val Leu Gly Leu Phe Thr Leu Ser Thr Gly Gln Asp 290 295 300 Ser Trp Asn Gln Thr Asn 305 310 <210> 22 <211> 310 <212> PRT <213> Artificial Sequence <220> <223> IgG degrading enzyme amino acid sequence <400> 22 Asp Ser Phe Ser Ala Asn Gln Glu Ile Arg Tyr Ser Glu Val Thr Pro 1 5 10 15 Tyr His Val Thr Ser Val Trp Thr Lys Gly Val Thr Pro Pro Ala Asn 20 25 30 Phe Thr Gln Gly Glu Asp Val Phe His Ala Pro Tyr Val Ala Asn Gln 35 40 45 Gly Trp Tyr Asp Ile Thr Lys Thr Phe Asn Gly Lys Asp Asp Leu Leu 50 55 60 Cys Gly Ala Ala Thr Ala Gly Asn Met Leu His Trp Trp Phe Asp Gln 65 70 75 80 Asn Lys Asp Gln Ile Glu Arg Tyr Leu Glu Glu His Pro Glu Lys Gln 85 90 95 Lys Ile Asn Phe Asn Gly Glu Gln Met Phe Asp Val Lys Glu Ala Ile 100 105 110 Asp Thr Lys Asn His Gln Leu Asp Ser Lys Leu Phe Glu Tyr Phe Lys 115 120 125 Glu Lys Ala Phe Pro Tyr Leu Ser Thr Lys His Leu Gly Val Phe Pro 130 135 140 Asp His Val Ile Asp Met Phe Ile Asn Gly Tyr Arg Leu Ser Leu Thr 145 150 155 160 Asn His Gly Pro Thr Pro Val Lys Glu Gly Ser Lys Asp Pro Arg Gly 165 170 175 Gly Ile Phe Asp Ala Val Phe Thr Arg Gly Asn Gln Ser Lys Leu Leu 180 185 190 Thr Ser Arg His Asp Phe Lys Glu Lys Asn Leu Lys Glu Ile Ser Asp 195 200 205 Leu Ile Lys Glu Glu Leu Thr Lys Gly Lys Ala Leu Gly Leu Ser His 210 215 220 Thr Tyr Ala Asn Val Arg Ile Asn His Val Ile Asn Leu Trp Gly Ala 225 230 235 240 Asp Phe Asp Ser Asn Gly Asn Leu Lys Ala Ile Tyr Val Thr Asp Ser 245 250 255 Asp Ser Asn Ala Ser Ile Gly Met Lys Lys Tyr Phe Val Gly Val Asn 260 265 270 Ser Ala Gly Lys Val Ala Ile Ser Ala Lys Glu Ile Lys Glu Lys Asn 275 280 285 Ile Gly Ala Gln Val Leu Gly Leu Phe Thr Leu Ser Thr Gly Gln Lys 290 295 300 Ser Trp Asn Gln Thr Asn 305 310 <210> 23 <211> 310 <212> PRT <213> Artificial Sequence <220> <223> IgG degrading enzyme amino acid sequence <400> 23 Asp Ser Phe Ser Ala Asn Gln Glu Ile Arg Tyr Ser Glu Val Thr Pro 1 5 10 15 Tyr His Val Thr Ser Val Trp Thr Lys Gly Val Thr Pro Pro Ala Asn 20 25 30 Phe Thr Gln Gly Glu Asp Val Phe His Ala Pro Tyr Val Ala Asn Gln 35 40 45 Gly Trp Tyr Asp Ile Thr Lys Thr Phe Asn Gly Lys Asp Asp Leu Leu 50 55 60 Cys Gly Ala Ala Thr Ala Gly Asn Met Leu His Trp Trp Phe Asp Gln 65 70 75 80 Asn Lys Asp Gln Ile Lys Arg Tyr Leu Lys Glu His Pro Glu Lys Gln 85 90 95 Lys Ile Asn Phe Arg Gly Glu Gln Met Phe Asp Val Lys Glu Ala Ile 100 105 110 Arg Thr Lys Asn His Gln Leu Asp Ser Lys Leu Phe Glu Tyr Phe Lys 115 120 125 Glu Lys Ala Phe Pro Tyr Leu Ser Thr Lys His Leu Gly Val Phe Pro 130 135 140 Asp His Val Ile Asp Met Phe Ile Asn Gly Tyr Arg Leu Ser Leu Thr 145 150 155 160 Asn His Gly Pro Thr Pro Val Lys Glu Gly Ser Lys Asp Pro Arg Gly 165 170 175 Gly Ile Phe Asp Ala Val Phe Thr Arg Gly Asn Gln Ser Lys Leu Leu 180 185 190 Thr Ser Arg His Asp Phe Lys Glu Lys Asn Leu Lys Glu Ile Ser Asp 195 200 205 Leu Ile Lys Ser Glu Leu Glu Asn Gly Lys Ala Leu Gly Leu Ser His 210 215 220 Thr Tyr Ala Asn Val Arg Ile Asn His Val Ile Asn Leu Trp Gly Ala 225 230 235 240 Asp Phe Asp Ser Asn Gly Asn Leu Lys Ala Ile Tyr Val Thr Asp Ser 245 250 255 Asp Ser Asn Ala Ser Ile Gly Met Lys Lys Tyr Phe Val Gly Val Asn 260 265 270 Lys Ala Gly Lys Val Ala Ile Ser Ala Lys Glu Ile Lys Glu Asp Asn 275 280 285 Ile Gly Ala Gln Val Leu Gly Leu Phe Thr Leu Ser Thr Gly Gln Asp 290 295 300 Ser Trp Asn Gln Thr Asn 305 310 <210> 24 <211> 310 <212> PRT <213> Artificial Sequence <220> <223> IgG degrading enzyme amino acid sequence <400> 24 Asp Ser Phe Ser Ala Asn Gln Glu Ile Arg Tyr Ser Glu Val Thr Pro 1 5 10 15 Tyr His Val Thr Ser Val Trp Thr Lys Gly Val Thr Pro Pro Ala Asn 20 25 30 Phe Thr Gln Gly Glu Asp Val Phe His Ala Pro Tyr Val Ala Asn Gln 35 40 45 Gly Trp Tyr Asp Ile Thr Lys Thr Phe Asn Gly Lys Asp Asp Leu Leu 50 55 60 Cys Gly Ala Ala Thr Ala Gly Asn Met Leu His Trp Trp Phe Asp Gln 65 70 75 80 Asn Lys Asp Gln Ile Lys Arg Tyr Leu Lys Glu His Pro Glu Lys Gln 85 90 95 Lys Ile Asn Phe Arg Gly Glu Gln Met Phe Asp Val Lys Glu Ala Ile 100 105 110 Arg Thr Lys Asn His Gln Leu Asp Ser Lys Leu Phe Glu Tyr Phe Lys 115 120 125 Glu Lys Ala Phe Pro Tyr Leu Ser Thr Lys His Leu Gly Val Phe Pro 130 135 140 Asp His Val Ile Asp Met Phe Ile Asn Gly Tyr Arg Leu Ser Leu Thr 145 150 155 160 Asn His Gly Pro Thr Pro Val Lys Lys Gly Ser Lys Asp Pro Arg Gly 165 170 175 Gly Ile Phe Asp Ala Val Phe Thr Arg Gly Asn Gln Ser Lys Leu Leu 180 185 190 Thr Ser Arg His Asp Phe Lys Glu Lys Asn Leu Lys Glu Ile Ser Asp 195 200 205 Leu Ile Lys Lys Glu Leu Glu Glu Gly Lys Ala Leu Gly Leu Ser His 210 215 220 Thr Tyr Ala Asn Val Arg Ile Asn His Val Ile Asn Leu Trp Gly Ala 225 230 235 240 Asp Phe Asp Ser Asn Gly Asn Leu Lys Ala Ile Tyr Val Thr Asp Ser 245 250 255 Asp Ser Asn Ala Ser Ile Gly Met Lys Lys Tyr Phe Val Gly Val Asn 260 265 270 Ser Ala Gly Lys Val Ala Ile Ser Ala Lys Glu Ile Lys Glu Asp Asn 275 280 285 Ile Gly Ala Gln Val Leu Gly Leu Phe Thr Leu Ser Thr Gly Gln Asp 290 295 300 Ser Trp Asn Gln Thr Asn 305 310 <210> 25 <211> 310 <212> PRT <213> Artificial Sequence <220> <223> IgG degrading enzyme amino acid sequence <400> 25 Asp Ser Phe Ser Ala Asn Gln Glu Ile Arg Tyr Ser Glu Val Thr Pro 1 5 10 15 Tyr His Val Thr Ser Val Trp Thr Lys Gly Val Thr Pro Pro Ala Asn 20 25 30 Phe Thr Gln Gly Glu Asp Val Phe His Ala Pro Tyr Val Ala Asn Gln 35 40 45 Gly Trp Tyr Asp Ile Thr Lys Thr Phe Asn Gly Lys Asp Asp Leu Leu 50 55 60 Cys Gly Ala Ala Thr Ala Gly Asn Met Leu His Trp Trp Phe Asp Gln 65 70 75 80 Asn Lys Asp Gln Ile Glu Arg Tyr Leu Glu Glu His Pro Glu Lys Gln 85 90 95 Lys Ile Asn Phe Arg Gly Glu Gln Met Phe Asp Val Lys Glu Ala Ile 100 105 110 Asp Thr Lys Asn His Gln Leu Asp Ser Lys Leu Phe Glu Tyr Phe Lys 115 120 125 Glu Lys Ala Phe Pro Tyr Leu Ser Thr Lys His Leu Gly Val Phe Pro 130 135 140 Asp His Val Ile Asp Met Phe Ile Asn Gly Tyr Arg Leu Ser Leu Thr 145 150 155 160 Asn His Gly Pro Thr Pro Val Lys Lys Gly Ser Lys Asp Pro Arg Gly 165 170 175 Gly Ile Phe Asp Ala Val Phe Thr Arg Gly Asn Gln Ser Lys Leu Leu 180 185 190 Thr Ser Arg His Asp Phe Lys Glu Lys Asn Leu Lys Glu Ile Ser Asp 195 200 205 Leu Ile Lys Glu Glu Leu Thr Lys Gly Lys Ala Leu Gly Leu Ser His 210 215 220 Thr Tyr Ala Asn Val Arg Ile Asn His Val Ile Asn Leu Trp Gly Ala 225 230 235 240 Asp Phe Asp Ser Asn Gly Asn Leu Lys Ala Ile Tyr Val Thr Asp Ser 245 250 255 Asp Ser Asn Ala Ser Ile Gly Met Lys Lys Tyr Phe Val Gly Val Asn 260 265 270 Ser Ala Gly Lys Val Ala Ile Ser Ala Lys Glu Ile Lys Glu Asp Asn 275 280 285 Ile Gly Ala Gln Val Leu Gly Leu Phe Thr Leu Ser Thr Gly Gln Lys 290 295 300 Ser Trp Asn Gln Thr Asn 305 310 <210> 26 <211> 306 <212> PRT <213> Artificial Sequence <220> <223> IgG degrading enzyme amino acid sequence <400> 26 Asp Ser Phe Ser Ala Asn Gln Glu Ile Arg Tyr Ser Glu Val Thr Pro 1 5 10 15 Tyr His Val Thr Ser Val Trp Thr Lys Gly Val Thr Pro Pro Ala Asn 20 25 30 Phe Thr Gln Gly Glu Asp Val Phe His Ala Pro Tyr Val Ala Asn Gln 35 40 45 Gly Trp Tyr Asp Ile Thr Lys Thr Phe Asn Gly Lys Asp Asp Leu Leu 50 55 60 Cys Gly Ala Ala Thr Ala Gly Asn Met Leu His Trp Trp Phe Asp Gln 65 70 75 80 Asn Lys Asp Gln Ile Lys Arg Tyr Leu Glu Glu His Pro Glu Lys Gln 85 90 95 Lys Ile Asn Phe Asn Gly Glu Gln Met Phe Asp Val Lys Glu Ala Ile 100 105 110 Asp Thr Lys Asn His Gln Leu Asp Ser Lys Leu Phe Glu Tyr Phe Lys 115 120 125 Glu Lys Ala Phe Pro Tyr Leu Ser Thr Lys His Leu Gly Val Phe Pro 130 135 140 Asp His Val Ile Asp Met Phe Ile Asn Gly Tyr Arg Leu Ser Leu Thr 145 150 155 160 Asn His Gly Pro Thr Pro Val Lys Glu Gly Ser Lys Asp Pro Arg Gly 165 170 175 Gly Ile Phe Asp Ala Val Phe Thr Arg Gly Asp Gln Ser Lys Leu Leu 180 185 190 Thr Ser Arg His Asp Phe Lys Glu Lys Asn Leu Lys Glu Ile Ser Asp 195 200 205 Leu Ile Lys Lys Glu Leu Thr Glu Gly Lys Ala Leu Gly Leu Ser His 210 215 220 Thr Tyr Ala Asn Val Arg Ile Asn His Val Ile Asn Leu Trp Gly Ala 225 230 235 240 Asp Phe Asp Ser Asn Gly Asn Leu Lys Ala Ile Tyr Val Thr Asp Ser 245 250 255 Asp Ser Asn Ala Ser Ile Gly Met Lys Lys Tyr Phe Val Gly Val Asn 260 265 270 Ser Ala Gly Lys Val Ala Ile Ser Ala Lys Glu Ile Lys Glu Asp Asn 275 280 285 Ile Gly Ala Gln Val Leu Gly Leu Phe Thr Leu Ser Thr Gly Gln Asp 290 295 300 Ser Trp 305 <210> 27 <211> 290 <212> PRT <213> Artificial Sequence <220> <223> IgG degrading enzyme amino acid sequence <400> 27 Ser Val Trp Thr Lys Gly Val Thr Pro Pro Ala Asn Phe Thr Gln Gly 1 5 10 15 Glu Asp Val Phe His Ala Pro Tyr Val Ala Asn Gin Gly Trp Tyr Asp 20 25 30 Ile Thr Lys Thr Phe Asn Gly Lys Asp Asp Leu Leu Cys Gly Ala Ala 35 40 45 Thr Ala Gly Asn Met Leu His Trp Trp Phe Asp Gln Asn Lys Asp Gln 50 55 60 Ile Lys Arg Tyr Leu Glu Glu His Pro Glu Lys Gln Lys Ile Asn Phe 65 70 75 80 Asn Gly Glu Gln Met Phe Asp Val Lys Glu Ala Ile Asp Thr Lys Asn 85 90 95 His Gln Leu Asp Ser Lys Leu Phe Glu Tyr Phe Lys Glu Lys Ala Phe 100 105 110 Pro Tyr Leu Ser Thr Lys His Leu Gly Val Phe Pro Asp His Val Ile 115 120 125 Asp Met Phe Ile Asn Gly Tyr Arg Leu Ser Leu Thr Asn His Gly Pro 130 135 140 Thr Pro Val Lys Glu Gly Ser Lys Asp Pro Arg Gly Gly Ile Phe Asp 145 150 155 160 Ala Val Phe Thr Arg Gly Asp Gln Ser Lys Leu Leu Thr Ser Arg His 165 170 175 Asp Phe Lys Glu Lys Asn Leu Lys Glu Ile Ser Asp Leu Ile Lys Lys 180 185 190 Glu Leu Thr Glu Gly Lys Ala Leu Gly Leu Ser His Thr Tyr Ala Asn 195 200 205 Val Arg Ile Asn His Val Ile Asn Leu Trp Gly Ala Asp Phe Asp Ser 210 215 220 Asn Gly Asn Leu Lys Ala Ile Tyr Val Thr Asp Ser Asp Ser Asn Ala 225 230 235 240 Ser Ile Gly Met Lys Lys Tyr Phe Val Gly Val Asn Ser Ala Gly Lys 245 250 255 Val Ala Ile Ser Ala Lys Glu Ile Lys Glu Asp Asn Ile Gly Ala Gln 260 265 270 Val Leu Gly Leu Phe Thr Leu Ser Thr Gly Gln Asp Ser Trp Asn Gln 275 280 285 Thr Asn 290 <210> 28 <211> 290 <212> PRT <213> Artificial Sequence <220> <223> IgG degrading enzyme amino acid sequence <400> 28 Ser Val Trp Thr Lys Gly Val Thr Pro Pro Ala Asn Phe Thr Gln Gly 1 5 10 15 Glu Asp Val Phe His Ala Pro Tyr Val Ala Asn Gin Gly Trp Tyr Asp 20 25 30 Ile Thr Lys Thr Phe Asn Gly Lys Asp Asp Leu Leu Cys Gly Ala Ala 35 40 45 Thr Ala Gly Asn Met Leu His Trp Trp Phe Asp Gln Asn Lys Asp Gln 50 55 60 Ile Lys Arg Tyr Leu Glu Glu His Pro Glu Lys Gln Lys Ile Asn Phe 65 70 75 80 Lys Gly Glu Gln Met Phe Asp Val Lys Glu Ala Ile Asp Thr Lys Asn 85 90 95 His Gln Leu Asp Ser Lys Leu Phe Glu Tyr Phe Lys Glu Lys Ala Phe 100 105 110 Pro Tyr Leu Ser Thr Lys His Leu Gly Val Phe Pro Asp His Val Ile 115 120 125 Asp Met Phe Ile Asn Gly Tyr Arg Leu Ser Leu Thr Asn His Gly Pro 130 135 140 Thr Pro Val Lys Glu Gly Ser Lys Asp Pro Arg Gly Gly Ile Phe Asp 145 150 155 160 Ala Val Phe Thr Arg Gly Asn Gln Ser Lys Leu Leu Thr Ser Arg His 165 170 175 Asp Phe Lys Glu Lys Asn Leu Lys Glu Ile Ser Asp Leu Ile Lys Lys 180 185 190 Glu Leu Thr Glu Gly Lys Ala Leu Gly Leu Ser His Thr Tyr Ala Asn 195 200 205 Val Arg Ile Asn His Val Ile Asn Leu Trp Gly Ala Asp Phe Asp Ser 210 215 220 Asn Gly Asn Leu Lys Ala Ile Tyr Val Thr Asp Ser Asp Ser Asn Ala 225 230 235 240 Ser Ile Gly Met Lys Lys Tyr Phe Val Gly Val Asn Ser Ala Gly Lys 245 250 255 Val Ala Ile Ser Ala Lys Glu Ile Lys Glu Asp Asn Ile Gly Ala Gln 260 265 270 Val Leu Gly Leu Phe Thr Leu Ser Thr Gly Gln Asp Ser Trp Asn Gln 275 280 285 Thr Asn 290 <210> 29 <211> 290 <212> PRT <213> Artificial Sequence <220> <223> IgG degrading enzyme amino acid sequence <400> 29 Ser Val Trp Thr Lys Gly Val Thr Pro Pro Ala Asn Phe Thr Gln Gly 1 5 10 15 Glu Asp Val Phe His Ala Pro Tyr Val Ala Asn Gin Gly Trp Tyr Asp 20 25 30 Ile Thr Lys Thr Phe Asn Gly Lys Asp Asp Leu Leu Cys Gly Ala Ala 35 40 45 Thr Ala Gly Asn Met Leu His Trp Trp Phe Asp Gln Asn Lys Asp Gln 50 55 60 Ile Glu Arg Tyr Leu Glu Glu His Pro Glu Lys Gln Lys Ile Asn Phe 65 70 75 80 Lys Gly Glu Gln Met Phe Asp Val Lys Lys Ala Ile Asp Thr Lys Asn 85 90 95 His Gln Leu Asp Ser Lys Leu Phe Glu Tyr Phe Lys Glu Lys Ala Phe 100 105 110 Pro Tyr Leu Ser Thr Lys His Leu Gly Val Phe Pro Asp His Val Ile 115 120 125 Asp Met Phe Ile Asn Gly Tyr Arg Leu Ser Leu Thr Asn His Gly Pro 130 135 140 Thr Pro Val Lys Glu Gly Ser Lys Asp Pro Arg Gly Gly Ile Phe Asp 145 150 155 160 Ala Val Phe Thr Arg Gly Asn Gln Ser Lys Leu Leu Thr Ser Arg His 165 170 175 Asp Phe Lys Glu Lys Asn Leu Lys Glu Ile Ser Asp Leu Ile Lys Glu 180 185 190 Glu Leu Thr Lys Gly Lys Ala Leu Gly Leu Ser His Thr Tyr Ala Asn 195 200 205 Val Arg Ile Asn His Val Ile Asn Leu Trp Gly Ala Asp Phe Asp Ser 210 215 220 Asn Gly Asn Leu Lys Ala Ile Tyr Val Thr Asp Ser Asp Ser Asn Ala 225 230 235 240 Ser Ile Gly Met Lys Lys Tyr Phe Val Gly Val Asn Ser Ala Gly Lys 245 250 255 Val Ala Ile Ser Ala Lys Glu Ile Lys Glu Asp Asn Ile Gly Ala Gln 260 265 270 Val Leu Gly Leu Phe Thr Leu Ser Thr Gly Gln Lys Ser Trp Asn Gln 275 280 285 Thr Asn 290 <210> 30 <211> 319 <212> PRT <213> Artificial Sequence <220> <223> IgG degrading enzyme amino acid sequence <400> 30 Asp Asp Tyr Gln Arg Asn Ala Thr Glu Ala Tyr Ala Lys Glu Val Pro 1 5 10 15 His Gln Ile Thr Ser Val Trp Thr Lys Gly Val Thr Pro Pro Ala Asn 20 25 30 Phe Thr Gln Gly Glu Asp Val Phe His Ala Pro Tyr Val Ala Asn Gln 35 40 45 Gly Trp Tyr Asp Ile Thr Lys Thr Phe Asn Gly Lys Asp Asp Leu Leu 50 55 60 Cys Gly Ala Ala Thr Ala Gly Asn Met Leu His Trp Trp Phe Asp Gln 65 70 75 80 Asn Lys Asp Gln Ile Glu Arg Tyr Leu Glu Glu His Pro Glu Lys Gln 85 90 95 Lys Ile Asn Phe Lys Gly Glu Gln Met Phe Asp Val Lys Lys Ala Ile 100 105 110 Asp Thr Lys Asn His Gln Leu Asp Ser Lys Leu Phe Glu Tyr Phe Lys 115 120 125 Glu Lys Ala Phe Pro Tyr Leu Ser Thr Lys His Leu Gly Val Phe Pro 130 135 140 Asp His Val Ile Asp Met Phe Ile Asn Gly Tyr Arg Leu Ser Leu Thr 145 150 155 160 Asn His Gly Pro Thr Pro Val Lys Glu Gly Ser Lys Asp Pro Arg Gly 165 170 175 Gly Ile Phe Asp Ala Val Phe Thr Arg Gly Asn Gln Ser Lys Leu Leu 180 185 190 Thr Ser Arg His Asp Phe Lys Glu Lys Asn Leu Lys Glu Ile Ser Asp 195 200 205 Leu Ile Lys Glu Glu Leu Thr Lys Gly Lys Ala Leu Gly Leu Ser His 210 215 220 Thr Tyr Ala Asn Val Arg Ile Asn His Val Ile Asn Leu Trp Gly Ala 225 230 235 240 Asp Phe Asp Ser Asn Gly Asn Leu Lys Ala Ile Tyr Val Thr Asp Ser 245 250 255 Asp Ser Asn Ala Ser Ile Gly Met Lys Lys Tyr Phe Val Gly Val Asn 260 265 270 Ser Ala Gly Lys Val Ala Ile Ser Ala Lys Glu Ile Lys Glu Asp Asn 275 280 285 Ile Gly Ala Gln Val Leu Gly Leu Phe Thr Leu Ser Thr Gly Gln Lys 290 295 300 Ser Trp Asn Gln Thr Asn Gly Gly Gly His His His His His His 305 310 315 <210> 31 <211> 315 <212> PRT <213> Artificial Sequence <220> <223> IgG degrading enzyme amino acid sequence <400> 31 Asp Asp Tyr Gln Arg Asn Ala Thr Glu Ala Tyr Ala Lys Glu Val Pro 1 5 10 15 His Gln Ile Thr Ser Val Trp Thr Lys Gly Val Thr Pro Leu Thr Pro 20 25 30 Glu Gln Phe Arg Tyr Asn Asn Glu Asp Val Ile His Ala Pro Tyr Leu 35 40 45 Ala His Gln Gly Trp Tyr Asp Ile Thr Lys Ala Phe Asp Gly Lys Asp 50 55 60 Asn Leu Leu Cys Gly Ala Ala Thr Ala Gly Asn Met Leu His Trp Trp 65 70 75 80 Phe Asp Gln Asn Lys Thr Glu Ile Glu Ala Tyr Leu Ser Lys His Pro 85 90 95 Glu Lys Gln Lys Ile Ile Phe Asn Asn Gln Glu Leu Phe Asp Leu Lys 100 105 110 Ala Ala Ile Asp Thr Lys Asp Ser Gln Thr Asn Ser Gln Leu Phe Asn 115 120 125 Tyr Phe Arg Asp Lys Ala Phe Pro Asn Leu Ser Ala Arg Gln Leu Gly 130 135 140 Val Met Pro Asp Leu Val Leu Asp Met Phe Ile Asn Gly Tyr Tyr Leu 145 150 155 160 Asn Val Phe Lys Thr Gln Ser Thr Asp Val Asn Arg Pro Tyr Gln Asp 165 170 175 Lys Asp Lys Arg Gly Gly Ile Phe Asp Ala Val Phe Thr Arg Gly Asp 180 185 190 Gln Thr Thr Leu Leu Thr Ala Arg His Asp Leu Lys Asn Lys Gly Leu 195 200 205 Asn Asp Ile Ser Thr Ile Ile Lys Gln Glu Leu Thr Glu Gly Arg Ala 210 215 220 Leu Ala Leu Ser His Thr Tyr Ala Asn Val Ser Ile Ser His Val Ile 225 230 235 240 Asn Leu Trp Gly Ala Asp Phe Asn Ala Glu Gly Asn Leu Glu Ala Ile 245 250 255 Tyr Val Thr Asp Ser Asp Ala Asn Ala Ser Ile Gly Met Lys Lys Tyr 260 265 270 Phe Val Gly Ile Asn Ala His Gly His Val Ala Ile Ser Ala Lys Lys 275 280 285 Ile Glu Gly Glu Asn Ile Gly Ala Gln Val Leu Gly Leu Phe Thr Leu 290 295 300 Ser Ser Gly Lys Asp Ile Trp Gln Lys Leu Ser 305 310 315 <210> 32 <211> 313 <212> PRT <213> Artificial Sequence <220> <223> IgG degrading enzyme amino acid sequence <400> 32 Asp Asp Tyr Gln Arg Asn Ala Thr Glu Ala Tyr Ala Lys Glu Val Pro 1 5 10 15 His Gln Ile Thr Ser Val Trp Thr Lys Gly Val Thr Pro Leu Thr Pro 20 25 30 Glu Gln Phe Arg Tyr Asn Asn Glu Asp Val Phe His Ala Pro Tyr Val 35 40 45 Ala Asn Gln Gly Trp Tyr Asp Ile Thr Lys Ala Phe Asp Gly Lys Asp 50 55 60 Asn Leu Leu Cys Gly Ala Ala Thr Ala Gly Asn Met Leu His Trp Trp 65 70 75 80 Phe Asp Gln Asn Lys Asp Gln Ile Lys Arg Tyr Leu Glu Glu His Pro 85 90 95 Glu Lys Gln Lys Ile Asn Phe Asn Gly Asp Asn Met Phe Asp Val Lys 100 105 110 Lys Ala Ile Asp Thr Lys Asn His Gln Leu Asp Ser Lys Leu Phe Asn 115 120 125 Tyr Phe Lys Glu Lys Ala Phe Pro Gly Leu Ser Ala Arg Arg Ile Gly 130 135 140 Val Phe Pro Asp His Val Ile Asp Met Phe Ile Asn Gly Tyr Arg Leu 145 150 155 160 Ser Leu Thr Asn His Gly Pro Thr Pro Val Lys Glu Gly Ser Lys Asp 165 170 175 Pro Arg Gly Gly Ile Phe Asp Ala Val Phe Thr Arg Gly Asn Gln Ser 180 185 190 Lys Leu Leu Thr Ser Arg His Asp Phe Lys Asn Lys Asn Leu Asn Asp 195 200 205 Ile Ser Thr Ile Ile Lys Gln Glu Leu Thr Lys Gly Lys Ala Leu Gly 210 215 220 Leu Ser His Thr Tyr Ala Asn Val Ser Ile Asn His Val Ile Asn Leu 225 230 235 240 Trp Gly Ala Asp Phe Asn Ala Glu Gly Asn Leu Glu Ala Ile Tyr Val 245 250 255 Thr Asp Ser Asp Ser Asn Ala Ser Ile Gly Met Lys Lys Tyr Phe Val 260 265 270 Gly Val Asn Ala His Gly His Val Ala Ile Ser Ala Lys Lys Ile Glu 275 280 285 Gly Glu Asn Ile Gly Ala Gln Val Leu Gly Leu Phe Thr Leu Ser Thr 290 295 300 Gly Gln Asp Ser Trp Gln Lys Leu Ser 305 310 <210> 33 <211> 313 <212> PRT <213> Artificial Sequence <220> <223> IgG degrading enzyme amino acid sequence <400> 33 Asp Asp Tyr Gln Arg Asn Ala Thr Glu Ala Tyr Ala Lys Glu Val Pro 1 5 10 15 His Gln Ile Thr Ser Val Trp Thr Lys Gly Val Thr Pro Leu Thr Pro 20 25 30 Glu Gln Phe Arg Tyr Asn Asn Glu Asp Val Ile His Ala Pro Tyr Leu 35 40 45 Ala Asn Gln Gly Trp Tyr Asp Ile Thr Lys Ala Phe Asp Gly Lys Asp 50 55 60 Asn Leu Leu Cys Gly Ala Ala Thr Ala Gly Asn Met Leu His Trp Trp 65 70 75 80 Phe Asp Gln Asn Lys Thr Glu Ile Glu Ala Tyr Leu Ser Lys His Pro 85 90 95 Glu Lys Gln Lys Ile Ile Phe Arg Asn Gln Glu Leu Phe Asp Leu Lys 100 105 110 Glu Ala Ile Arg Thr Lys Asp Ser Gln Thr Gln Leu Phe Glu Tyr Phe 115 120 125 Arg Asp Lys Ala Phe Pro Tyr Leu Ser Ala Arg Gln Leu Gly Val Met 130 135 140 Pro Asp Leu Val Leu Asp Met Phe Ile Asn Gly Tyr Tyr Leu Asn Val 145 150 155 160 Phe Lys Thr Gln Ser Thr Asp Val Lys Arg Pro Tyr Gln Asp Lys Asp 165 170 175 Lys Arg Gly Gly Ile Phe Asp Ala Val Phe Thr Arg Gly Asn Gln Thr 180 185 190 Thr Leu Leu Thr Ala Arg His Asp Leu Lys Asn Lys Gly Leu Asn Asp 195 200 205 Ile Ser Thr Ile Ile Lys Glu Glu Leu Thr Lys Gly Arg Ala Leu Ala 210 215 220 Leu Ser His Thr Tyr Ala Asn Val Ser Ile Ser His Val Ile Asn Leu 225 230 235 240 Trp Gly Ala Asp Phe Asn Ala Glu Gly Asn Leu Glu Ala Thr Tyr Val 245 250 255 Thr Asp Ser Asp Ala Asn Ala Ser Ile Gly Met Lys Lys Tyr Phe Val 260 265 270 Gly Ile Asn Lys His Gly His Val Ala Ile Ser Ala Lys Lys Ile Glu 275 280 285 Gly Glu Asn Ile Gly Ala Gln Val Leu Gly Leu Phe Thr Leu Ser Ser 290 295 300 Gly Lys Asp Ile Trp Gln Lys Leu Asn 305 310 <210> 34 <211> 315 <212> PRT <213> Artificial Sequence <220> <223> IgG degrading enzyme amino acid sequence <400> 34 Asp Asp Tyr Gln Arg Asn Ala Thr Glu Ala Tyr Ala Lys Glu Val Pro 1 5 10 15 His Gln Ile Thr Ser Val Trp Thr Lys Gly Val Thr Pro Leu Thr Pro 20 25 30 Glu Gln Phe Arg Tyr Asn Asn Glu Asp Val Ile His Ala Pro Tyr Leu 35 40 45 Ala His Gln Gly Trp Tyr Asp Ile Thr Lys Thr Phe Asn Gly Lys Asp 50 55 60 Asn Leu Leu Cys Gly Ala Ala Thr Ala Gly Asn Met Leu His Trp Trp 65 70 75 80 Phe Asp Gln Asn Lys Thr Glu Ile Glu Ala Tyr Leu Ser Lys His Pro 85 90 95 Glu Lys Gln Lys Ile Ile Phe Asn Asn Glu Glu Leu Phe Asp Leu Lys 100 105 110 Ala Ala Ile Asp Thr Lys Asp Ser Gln Thr Asn Ser Gln Leu Phe Asn 115 120 125 Tyr Phe Lys Glu Lys Ala Phe Pro Asn Leu Ser Thr Arg Gln Leu Gly 130 135 140 Val Met Pro Asp Leu Val Leu Asp Met Phe Ile Asn Gly Tyr Tyr Leu 145 150 155 160 Asn Val Phe Lys Thr Gln Ser Thr Asp Val Asn Arg Pro Tyr Gln Asp 165 170 175 Lys Asp Lys Arg Gly Gly Ile Phe Asp Ala Val Phe Thr Arg Gly Asn 180 185 190 Gln Thr Thr Leu Leu Thr Ala Arg His Asp Phe Lys Glu Lys Gly Leu 195 200 205 Lys Asp Ile Ser Thr Ile Ile Lys Gln Glu Leu Thr Glu Gly Arg Ala 210 215 220 Leu Ala Leu Ser His Thr Tyr Ala Asn Val Ser Ile Ser His Val Ile 225 230 235 240 Asn Leu Trp Gly Ala Asp Phe Asp Ala Glu Gly Asn Leu Lys Ala Ile 245 250 255 Tyr Val Thr Asp Ser Asp Ala Asn Ala Ser Ile Gly Met Lys Lys Tyr 260 265 270 Phe Val Gly Ile Asn Ala His Gly Lys Val Ala Ile Ser Ala Lys Lys 275 280 285 Ile Glu Gly Glu Asn Ile Gly Ala Gln Val Leu Gly Leu Phe Thr Leu 290 295 300 Ser Ser Gly Lys Asp Ile Trp Gln Gln Leu Ser 305 310 315 <210> 35 <211> 315 <212> PRT <213> Artificial Sequence <220> <223> IgG degrading enzyme amino acid sequence <400> 35 Asp Ser Phe Ser Ala Asn Gln Glu Ile Arg Tyr Ser Glu Val Thr Pro 1 5 10 15 Tyr His Val Thr Ser Val Trp Thr Lys Gly Val Thr Pro Leu Thr Pro 20 25 30 Glu Gln Phe Arg Tyr Asn Asn Glu Asp Val Ile His Ala Pro Tyr Leu 35 40 45 Ala His Gln Gly Trp Tyr Asp Ile Thr Lys Ala Phe Asp Gly Lys Asp 50 55 60 Asn Leu Leu Cys Gly Ala Ala Thr Ala Gly Asn Met Leu His Trp Trp 65 70 75 80 Phe Asp Gln Asn Lys Thr Glu Ile Glu Ala Tyr Leu Ser Lys His Pro 85 90 95 Glu Lys Gln Lys Ile Ile Phe Asn Asn Gln Glu Leu Phe Asp Leu Lys 100 105 110 Ala Ala Ile Asp Thr Lys Asp Ser Gln Thr Asn Ser Gln Leu Phe Asn 115 120 125 Tyr Phe Arg Asp Lys Ala Phe Pro Asn Leu Ser Ala Arg Gln Leu Gly 130 135 140 Val Met Pro Asp Leu Val Leu Asp Met Phe Ile Asn Gly Tyr Tyr Leu 145 150 155 160 Asn Val Phe Lys Thr Gln Ser Thr Asp Val Asn Arg Pro Tyr Gln Asp 165 170 175 Lys Asp Lys Arg Gly Gly Ile Phe Asp Ala Val Phe Thr Arg Gly Asp 180 185 190 Gln Thr Thr Leu Leu Thr Ala Arg His Asp Leu Lys Asn Lys Gly Leu 195 200 205 Asn Asp Ile Ser Thr Ile Ile Lys Gln Glu Leu Thr Glu Gly Arg Ala 210 215 220 Leu Ala Leu Ser His Thr Tyr Ala Asn Val Ser Ile Ser His Val Ile 225 230 235 240 Asn Leu Trp Gly Ala Asp Phe Asn Ala Glu Gly Asn Leu Glu Ala Ile 245 250 255 Tyr Val Thr Asp Ser Asp Ala Asn Ala Ser Ile Gly Met Lys Lys Tyr 260 265 270 Phe Val Gly Ile Asn Ala His Gly His Val Ala Ile Ser Ala Lys Lys 275 280 285 Ile Glu Gly Glu Asn Ile Gly Ala Gln Val Leu Gly Leu Phe Thr Leu 290 295 300 Ser Ser Gly Lys Asp Ile Trp Gln Lys Leu Ser 305 310 315 <210> 36 <211> 295 <212> PRT <213> Artificial Sequence <220> <223> IgG degrading enzyme amino acid sequence <400> 36 Ser Val Trp Thr Lys Gly Val Thr Pro Leu Thr Pro Glu Gln Phe Arg 1 5 10 15 Tyr Asn Asn Glu Asp Val Ile His Ala Pro Tyr Leu Ala His Gln Gly 20 25 30 Trp Tyr Asp Ile Thr Lys Ala Phe Asp Gly Lys Asp Asn Leu Leu Cys 35 40 45 Gly Ala Ala Thr Ala Gly Asn Met Leu His Trp Trp Phe Asp Gln Asn 50 55 60 Lys Thr Glu Ile Glu Ala Tyr Leu Ser Lys His Pro Glu Lys Gln Lys 65 70 75 80 Ile Ile Phe Asn Asn Gln Glu Leu Phe Asp Leu Lys Ala Ala Ile Asp 85 90 95 Thr Lys Asp Ser Gln Thr Asn Ser Gln Leu Phe Asn Tyr Phe Arg Asp 100 105 110 Lys Ala Phe Pro Asn Leu Ser Ala Arg Gln Leu Gly Val Met Pro Asp 115 120 125 Leu Val Leu Asp Met Phe Ile Asn Gly Tyr Tyr Leu Asn Val Phe Lys 130 135 140 Thr Gln Ser Thr Asp Val Asn Arg Pro Tyr Gln Asp Lys Asp Lys Arg 145 150 155 160 Gly Gly Ile Phe Asp Ala Val Phe Thr Arg Gly Asp Gln Thr Thr Leu 165 170 175 Leu Thr Ala Arg His Asp Leu Lys Asn Lys Gly Leu Asn Asp Ile Ser 180 185 190 Thr Ile Ile Lys Gln Glu Leu Thr Glu Gly Arg Ala Leu Ala Leu Ser 195 200 205 His Thr Tyr Ala Asn Val Ser Ile Ser His Val Ile Asn Leu Trp Gly 210 215 220 Ala Asp Phe Asn Ala Glu Gly Asn Leu Glu Ala Ile Tyr Val Thr Asp 225 230 235 240 Ser Asp Ala Asn Ala Ser Ile Gly Met Lys Lys Tyr Phe Val Gly Ile 245 250 255 Asn Ala His Gly His Val Ala Ile Ser Ala Lys Lys Ile Glu Gly Glu 260 265 270 Asn Ile Gly Ala Gln Val Leu Gly Leu Phe Thr Leu Ser Ser Gly Lys 275 280 285 Asp Ile Trp Gln Lys Leu Ser 290 295 <210> 37 <211> 314 <212> PRT <213> Artificial Sequence <220> <223> IgG degrading enzyme amino acid sequence <400> 37 Asp Asp Tyr Gln Arg Asn Ala Thr Glu Ala Tyr Ala Lys Glu Val Pro 1 5 10 15 His Gln Ile Thr Ser Val Trp Thr Lys Gly Val Thr Pro Leu Thr Pro 20 25 30 Glu Gln Phe Thr Gln Gly Glu Asp Val Ile His Ala Pro Tyr Leu Ala 35 40 45 His Gln Gly Trp Tyr Asp Ile Thr Lys Ala Phe Asp Gly Lys Asp Asn 50 55 60 Leu Leu Cys Gly Ala Ala Thr Ala Gly Asn Met Leu His Trp Trp Phe 65 70 75 80 Asp Gln Asn Lys Thr Glu Ile Glu Ala Tyr Leu Ser Lys His Pro Glu 85 90 95 Lys Gln Lys Ile Ile Phe Asn Asn Gln Glu Leu Phe Asp Leu Lys Ala 100 105 110 Ala Ile Asp Thr Lys Asp Ser Gln Thr Asn Ser Gln Leu Phe Asn Tyr 115 120 125 Phe Arg Asp Lys Ala Phe Pro Asn Leu Ser Ala Arg Gln Leu Gly Val 130 135 140 Met Pro Asp Leu Val Leu Asp Met Phe Ile Asn Gly Tyr Tyr Leu Asn 145 150 155 160 Val Phe Lys Thr Gln Ser Thr Asp Val Asn Arg Pro Tyr Gln Asp Lys 165 170 175 Asp Lys Arg Gly Gly Ile Phe Asp Ala Val Phe Thr Arg Gly Asp Gln 180 185 190 Thr Thr Leu Leu Thr Ala Arg His Asp Leu Lys Asn Lys Gly Leu Asn 195 200 205 Asp Ile Ser Thr Ile Ile Lys Gln Glu Leu Thr Glu Gly Arg Ala Leu 210 215 220 Ala Leu Ser His Thr Tyr Ala Asn Val Ser Ile Ser His Val Ile Asn 225 230 235 240 Leu Trp Gly Ala Asp Phe Asn Ala Glu Gly Asn Leu Glu Ala Ile Tyr 245 250 255 Val Thr Asp Ser Asp Ala Asn Ala Ser Ile Gly Met Lys Lys Tyr Phe 260 265 270 Val Gly Ile Asn Ala His Gly His Val Ala Ile Ser Ala Lys Lys Ile 275 280 285 Glu Gly Glu Asn Ile Gly Ala Gln Val Leu Gly Leu Phe Thr Leu Ser 290 295 300 Ser Gly Lys Asp Ile Trp Gln Lys Leu Ser 305 310 <210> 38 <211> 312 <212> PRT <213> Artificial Sequence <220> <223> IgG degrading enzyme amino acid sequence <400> 38 Asp Asp Tyr Gln Arg Asn Ala Thr Glu Ala Tyr Ala Lys Glu Val Pro 1 5 10 15 His Gln Ile Thr Ser Val Trp Thr Lys Gly Val Thr Pro Glu Gln 20 25 30 Phe Thr Gln Gly Glu Asp Val Ile His Ala Pro Tyr Leu Ala His Gln 35 40 45 Gly Trp Tyr Asp Ile Thr Lys Ala Phe Asp Gly Lys Asp Asn Leu Leu 50 55 60 Cys Gly Ala Ala Thr Ala Gly Asn Met Leu His Trp Trp Phe Asp Gln 65 70 75 80 Asn Lys Thr Glu Ile Glu Ala Tyr Leu Ser Lys His Pro Glu Lys Gln 85 90 95 Lys Ile Ile Phe Asn Asn Gln Glu Leu Phe Asp Leu Lys Ala Ala Ile 100 105 110 Asp Thr Lys Asp Ser Gln Thr Asn Ser Gln Leu Phe Asn Tyr Phe Arg 115 120 125 Asp Lys Ala Phe Pro Asn Leu Ser Ala Arg Gln Leu Gly Val Met Pro 130 135 140 Asp Leu Val Leu Asp Met Phe Ile Asn Gly Tyr Tyr Leu Asn Val Phe 145 150 155 160 Lys Thr Gln Ser Thr Asp Val Asn Arg Pro Tyr Gln Asp Lys Asp Lys 165 170 175 Arg Gly Gly Ile Phe Asp Ala Val Phe Thr Arg Gly Asp Gln Thr Thr 180 185 190 Leu Leu Thr Ala Arg His Asp Leu Lys Asn Lys Gly Leu Asn Asp Ile 195 200 205 Ser Thr Ile Ile Lys Gln Glu Leu Thr Glu Gly Arg Ala Leu Ala Leu 210 215 220 Ser His Thr Tyr Ala Asn Val Ser Ile Ser His Val Ile Asn Leu Trp 225 230 235 240 Gly Ala Asp Phe Asn Ala Glu Gly Asn Leu Glu Ala Ile Tyr Val Thr 245 250 255 Asp Ser Asp Ala Asn Ala Ser Ile Gly Met Lys Lys Tyr Phe Val Gly 260 265 270 Ile Asn Ala His Gly His Val Ala Ile Ser Ala Lys Lys Ile Glu Gly 275 280 285 Glu Asn Ile Gly Ala Gln Val Leu Gly Leu Phe Thr Leu Ser Ser Gly 290 295 300 Lys Asp Ile Trp Gln Lys Leu Ser 305 310 <210> 39 <211> 313 <212> PRT <213> Artificial Sequence <220> <223> IgG degrading enzyme amino acid sequence <400> 39 Asp Asp Tyr Gln Arg Asn Ala Thr Glu Ala Tyr Ala Lys Glu Val Pro 1 5 10 15 His Gln Ile Thr Ser Val Trp Thr Lys Gly Val Thr Pro Glu Gln 20 25 30 Phe Arg Tyr Asn Asn Glu Asp Val Ile His Ala Pro Tyr Leu Ala His 35 40 45 Gln Gly Trp Tyr Asp Ile Thr Lys Ala Phe Asp Gly Lys Asp Asn Leu 50 55 60 Leu Cys Gly Ala Ala Thr Ala Gly Asn Met Leu His Trp Trp Phe Asp 65 70 75 80 Gln Asn Lys Thr Glu Ile Glu Ala Tyr Leu Ser Lys His Pro Glu Lys 85 90 95 Gln Lys Ile Ile Phe Asn Asn Gln Glu Leu Phe Asp Leu Lys Ala Ala 100 105 110 Ile Asp Thr Lys Asp Ser Gln Thr Asn Ser Gln Leu Phe Asn Tyr Phe 115 120 125 Arg Asp Lys Ala Phe Pro Asn Leu Ser Ala Arg Gln Leu Gly Val Met 130 135 140 Pro Asp Leu Val Leu Asp Met Phe Ile Asn Gly Tyr Tyr Leu Asn Val 145 150 155 160 Phe Lys Thr Gln Ser Thr Asp Val Asn Arg Pro Tyr Gln Asp Lys Asp 165 170 175 Lys Arg Gly Gly Ile Phe Asp Ala Val Phe Thr Arg Gly Asp Gln Thr 180 185 190 Thr Leu Leu Thr Ala Arg His Asp Leu Lys Asn Lys Gly Leu Asn Asp 195 200 205 Ile Ser Thr Ile Ile Lys Gln Glu Leu Thr Glu Gly Arg Ala Leu Ala 210 215 220 Leu Ser His Thr Tyr Ala Asn Val Ser Ile Ser His Val Ile Asn Leu 225 230 235 240 Trp Gly Ala Asp Phe Asn Ala Glu Gly Asn Leu Glu Ala Ile Tyr Val 245 250 255 Thr Asp Ser Asp Ala Asn Ala Ser Ile Gly Met Lys Lys Tyr Phe Val 260 265 270 Gly Ile Asn Ala His Gly His Val Ala Ile Ser Ala Lys Lys Ile Glu 275 280 285 Gly Glu Asn Ile Gly Ala Gln Val Leu Gly Leu Phe Thr Leu Ser Ser 290 295 300 Gly Lys Asp Ile Trp Gln Lys Leu Ser 305 310 <210> 40 <211> 312 <212> PRT <213> Artificial Sequence <220> <223> IgG degrading enzyme amino acid sequence <400> 40 Asp Asp Tyr Gln Arg Asn Ala Thr Glu Ala Tyr Ala Lys Glu Val Pro 1 5 10 15 His Gln Ile Thr Ser Val Trp Thr Lys Gly Val Thr Pro Glu Gln 20 25 30 Phe Thr Gln Gly Glu Asp Val Ile His Ala Pro Tyr Leu Ala His Gln 35 40 45 Gly Trp Tyr Asp Ile Thr Lys Ala Phe Asp Gly Lys Asp Asn Leu Leu 50 55 60 Cys Gly Ala Ala Thr Ala Gly Asn Met Leu His Trp Trp Phe Asp Gln 65 70 75 80 Asn Lys Thr Glu Ile Glu Ala Tyr Leu Ser Lys His Pro Glu Lys Gln 85 90 95 Lys Ile Ile Ile Asn Asn Gln Glu Leu Phe Asp Leu Lys Ala Ala Ile 100 105 110 Asp Thr Lys Asp Ser Gln Thr Asn Ser Gln Leu Phe Asn Tyr Phe Arg 115 120 125 Asp Lys Ala Phe Pro Asn Leu Ser Ala Arg Gln Leu Gly Val Met Pro 130 135 140 Asp Leu Val Leu Asp Met Phe Ile Asn Gly Tyr Tyr Leu Asn Val Phe 145 150 155 160 Lys Thr Gln Ser Thr Asp Val Asn Arg Pro Tyr Gln Asp Lys Asp Lys 165 170 175 Arg Gly Gly Ile Phe Asp Ala Val Phe Thr Arg Gly Asp Gln Thr Thr 180 185 190 Leu Leu Thr Ala Arg His Asp Leu Lys Asn Lys Gly Leu Asn Asp Ile 195 200 205 Ser Thr Ile Ile Lys Gln Glu Leu Thr Glu Gly Arg Ala Leu Ala Leu 210 215 220 Ser His Thr Tyr Ala Asn Val Ser Ile Ser His Val Ile Asn Leu Trp 225 230 235 240 Gly Ala Asp Phe Asn Ala Glu Gly Asn Leu Glu Ala Ile Tyr Val Thr 245 250 255 Asp Ser Asp Ala Asn Ala Ser Ile Gly Met Lys Lys Tyr Phe Val Gly 260 265 270 Ile Asn Ala His Gly His Val Ala Ile Ser Ala Lys Lys Ile Glu Gly 275 280 285 Glu Asn Ile Gly Ala Gln Val Leu Gly Leu Phe Thr Leu Ser Ser Gly 290 295 300 Lys Asp Ile Trp Gln Lys Leu Ser 305 310 <210> 41 <211> 312 <212> PRT <213> Artificial Sequence <220> <223> IgG degrading enzyme amino acid sequence <400> 41 Asp Asp Tyr Gln Arg Asn Ala Thr Glu Ala Tyr Ala Lys Glu Val Pro 1 5 10 15 His Gln Ile Thr Ser Val Trp Thr Lys Gly Val Thr Pro Glu Gln 20 25 30 Phe Thr Gln Gly Glu Asp Val Ile His Ala Pro Tyr Leu Ala His Gln 35 40 45 Gly Trp Tyr Asp Ile Thr Lys Ala Phe Asp Gly Lys Asp Asn Leu Leu 50 55 60 Cys Gly Ala Ala Thr Ala Gly Asn Met Leu His Trp Trp Phe Asp Gln 65 70 75 80 Asn Lys Thr Glu Ile Glu Ala Tyr Leu Ser Lys His Pro Glu Lys Gln 85 90 95 Lys Ile Ile Phe Arg Asn Gln Glu Leu Phe Asp Leu Lys Ala Ala Ile 100 105 110 Asp Thr Lys Asp Ser Gln Thr Asn Ser Gln Leu Phe Asn Tyr Phe Arg 115 120 125 Asp Lys Ala Phe Pro Asn Leu Ser Ala Arg Gln Leu Gly Val Met Pro 130 135 140 Asp Leu Val Leu Asp Met Phe Ile Asn Gly Tyr Tyr Leu Asn Val Phe 145 150 155 160 Lys Thr Gln Ser Thr Asp Val Asn Arg Pro Tyr Gln Asp Lys Asp Lys 165 170 175 Arg Gly Gly Ile Phe Asp Ala Val Phe Thr Arg Gly Asp Gln Thr Thr 180 185 190 Leu Leu Thr Ala Arg His Asp Leu Lys Asn Lys Gly Leu Asn Asp Ile 195 200 205 Ser Thr Ile Ile Lys Gln Glu Leu Thr Glu Gly Arg Ala Leu Ala Leu 210 215 220 Ser His Thr Tyr Ala Asn Val Ser Ile Ser His Val Ile Asn Leu Trp 225 230 235 240 Gly Ala Asp Phe Asn Ala Glu Gly Asn Leu Glu Ala Ile Tyr Val Thr 245 250 255 Asp Ser Asp Ala Asn Ala Ser Ile Gly Met Lys Lys Tyr Phe Val Gly 260 265 270 Ile Asn Ala His Gly His Val Ala Ile Ser Ala Lys Lys Ile Glu Gly 275 280 285 Glu Asn Ile Gly Ala Gln Val Leu Gly Leu Phe Thr Leu Ser Ser Gly 290 295 300 Lys Asp Ile Trp Gln Lys Leu Ser 305 310 <210> 42 <211> 312 <212> PRT <213> Artificial Sequence <220> <223> IgG degrading enzyme amino acid sequence <400> 42 Asp Asp Tyr Gln Arg Asn Ala Thr Glu Ala Tyr Ala Lys Glu Val Pro 1 5 10 15 His Gln Ile Thr Ser Val Trp Thr Lys Gly Val Thr Pro Glu Gln 20 25 30 Phe Thr Gln Gly Glu Asp Val Ile His Ala Pro Tyr Leu Ala His Gln 35 40 45 Gly Trp Tyr Asp Ile Thr Lys Ala Phe Asp Gly Lys Asp Asn Leu Leu 50 55 60 Cys Gly Ala Ala Thr Ala Gly Asn Met Leu His Trp Trp Phe Asp Gln 65 70 75 80 Asn Lys Thr Glu Ile Glu Ala Tyr Leu Ser Lys His Pro Glu Lys Gln 85 90 95 Lys Ile Ile Ile Arg Asn Gln Glu Leu Phe Asp Leu Lys Ala Ala Ile 100 105 110 Asp Thr Lys Asp Ser Gln Thr Asn Ser Gln Leu Phe Asn Tyr Phe Arg 115 120 125 Asp Lys Ala Phe Pro Asn Leu Ser Ala Arg Gln Leu Gly Val Met Pro 130 135 140 Asp Leu Val Leu Asp Met Phe Ile Asn Gly Tyr Tyr Leu Asn Val Phe 145 150 155 160 Lys Thr Gln Ser Thr Asp Val Asn Arg Pro Tyr Gln Asp Lys Asp Lys 165 170 175 Arg Gly Gly Ile Phe Asp Ala Val Phe Thr Arg Gly Asp Gln Thr Thr 180 185 190 Leu Leu Thr Ala Arg His Asp Leu Lys Asn Lys Gly Leu Asn Asp Ile 195 200 205 Ser Thr Ile Ile Lys Gln Glu Leu Thr Glu Gly Arg Ala Leu Ala Leu 210 215 220 Ser His Thr Tyr Ala Asn Val Ser Ile Ser His Val Ile Asn Leu Trp 225 230 235 240 Gly Ala Asp Phe Asn Ala Glu Gly Asn Leu Glu Ala Ile Tyr Val Thr 245 250 255 Asp Ser Asp Ala Asn Ala Ser Ile Gly Met Lys Lys Tyr Phe Val Gly 260 265 270 Ile Asn Ala His Gly His Val Ala Ile Ser Ala Lys Lys Ile Glu Gly 275 280 285 Glu Asn Ile Gly Ala Gln Val Leu Gly Leu Phe Thr Leu Ser Ser Gly 290 295 300 Lys Asp Ile Trp Gln Lys Leu Ser 305 310 <210> 43 <211> 312 <212> PRT <213> Artificial Sequence <220> <223> IgG degrading enzyme amino acid sequence <400> 43 Asp Asp Tyr Gln Arg Asn Ala Thr Glu Ala Tyr Ala Lys Glu Val Pro 1 5 10 15 His Gln Ile Thr Ser Val Trp Thr Lys Gly Val Thr Pro Glu Gln 20 25 30 Phe Thr Gln Gly Glu Asp Val Ile His Ala Pro Tyr Leu Ala Asn Gln 35 40 45 Gly Trp Tyr Asp Ile Thr Lys Ala Phe Asp Gly Lys Asp Asn Leu Leu 50 55 60 Cys Gly Ala Ala Thr Ala Gly Asn Met Leu His Trp Trp Phe Asp Gln 65 70 75 80 Asn Lys Thr Glu Ile Glu Ala Tyr Leu Ser Lys His Pro Glu Lys Gln 85 90 95 Lys Ile Ile Phe Arg Asn Gln Glu Leu Phe Asp Leu Lys Glu Ala Ile 100 105 110 Arg Thr Lys Asp Ser Gln Thr Asn Ser Gln Leu Phe Glu Tyr Phe Arg 115 120 125 Asp Lys Ala Phe Pro Tyr Leu Ser Ala Arg Gln Leu Gly Val Met Pro 130 135 140 Asp Leu Val Leu Asp Met Phe Ile Asn Gly Tyr Tyr Leu Asn Val Phe 145 150 155 160 Lys Thr Gln Ser Thr Asp Val Lys Arg Pro Tyr Gln Asp Lys Asp Lys 165 170 175 Arg Gly Gly Ile Phe Asp Ala Val Phe Thr Arg Gly Asn Gln Thr Thr 180 185 190 Leu Leu Thr Ala Arg His Asp Leu Lys Asn Lys Gly Leu Asn Asp Ile 195 200 205 Ser Thr Ile Ile Lys Glu Glu Leu Thr Lys Gly Arg Ala Leu Ala Leu 210 215 220 Ser His Thr Tyr Ala Asn Val Ser Ile Ser His Val Ile Asn Leu Trp 225 230 235 240 Gly Ala Asp Phe Asn Ala Glu Gly Asn Leu Glu Ala Ile Tyr Val Thr 245 250 255 Asp Ser Asp Ala Asn Ala Ser Ile Gly Met Lys Lys Tyr Phe Val Gly 260 265 270 Ile Asn Lys His Gly His Val Ala Ile Ser Ala Lys Lys Ile Glu Gly 275 280 285 Glu Asn Ile Gly Ala Gln Val Leu Gly Leu Phe Thr Leu Ser Ser Gly 290 295 300 Lys Asp Ile Trp Gln Lys Leu Asn 305 310 <210> 44 <211> 292 <212> PRT <213> Artificial Sequence <220> <223> IgG degrading enzyme amino acid sequence <400> 44 Ser Val Trp Thr Lys Gly Val Thr Pro Pro Glu Gln Phe Thr Gln Gly 1 5 10 15 Glu Asp Val Ile His Ala Pro Tyr Leu Ala His Gln Gly Trp Tyr Asp 20 25 30 Ile Thr Lys Ala Phe Asp Gly Lys Asp Asn Leu Leu Cys Gly Ala Ala 35 40 45 Thr Ala Gly Asn Met Leu His Trp Trp Phe Asp Gln Asn Lys Thr Glu 50 55 60 Ile Glu Ala Tyr Leu Ser Lys His Pro Glu Lys Gln Lys Ile Ile Phe 65 70 75 80 Arg Asn Gln Glu Leu Phe Asp Leu Lys Ala Ala Ile Asp Thr Lys Asp 85 90 95 Ser Gln Thr Asn Ser Gln Leu Phe Asn Tyr Phe Arg Asp Lys Ala Phe 100 105 110 Pro Asn Leu Ser Ala Arg Gln Leu Gly Val Met Pro Asp Leu Val Leu 115 120 125 Asp Met Phe Ile Asn Gly Tyr Tyr Leu Asn Val Phe Lys Thr Gln Ser 130 135 140 Thr Asp Val Asn Arg Pro Tyr Gln Asp Lys Asp Lys Arg Gly Gly Ile 145 150 155 160 Phe Asp Ala Val Phe Thr Arg Gly Asn Gln Thr Thr Leu Leu Thr Ala 165 170 175 Arg His Asp Leu Lys Asn Lys Gly Leu Asn Asp Ile Ser Thr Ile Ile 180 185 190 Lys Gln Glu Leu Thr Glu Gly Arg Ala Leu Ala Leu Ser His Thr Tyr 195 200 205 Ala Asn Val Ser Ile Ser His Val Ile Asn Leu Trp Gly Ala Asp Phe 210 215 220 Asn Ala Glu Gly Asn Leu Glu Ala Thr Tyr Val Thr Asp Ser Asp Ala 225 230 235 240 Asn Ala Ser Ile Gly Met Lys Lys Tyr Phe Val Gly Ile Asn Ala His 245 250 255 Gly His Val Ala Ile Ser Ala Lys Lys Ile Glu Gly Glu Asn Ile Gly 260 265 270 Ala Gln Val Leu Gly Leu Phe Thr Leu Ser Ser Gly Lys Asp Ile Trp 275 280 285 Gln Lys Leu Ser 290 <210> 45 <211> 312 <212> PRT <213> Artificial Sequence <220> <223> IgG degrading enzyme amino acid sequence <400> 45 Asp Asp Tyr Gln Arg Asn Ala Thr Glu Ala Tyr Ala Lys Glu Val Pro 1 5 10 15 His Gln Ile Thr Ser Val Trp Thr Lys Gly Val Thr Pro Glu Gln 20 25 30 Phe Thr Gln Gly Glu Asp Val Ile His Ala Pro Tyr Leu Ala His Gln 35 40 45 Gly Trp Tyr Asp Ile Thr Lys Ala Phe Asp Gly Ala Asp Asn Leu Leu 50 55 60 Cys Gly Ala Ala Thr Ala Gly Asn Met Leu His Trp Trp Phe Asp Gln 65 70 75 80 Asn Lys Thr Glu Ile Glu Ala Tyr Leu Ser Lys His Pro Glu Lys Gln 85 90 95 Lys Ile Ile Phe Arg Asn Gln Glu Leu Phe Asp Leu Lys Ala Ala Ile 100 105 110 Asp Thr Lys Asp Ser Gln Thr Asn Ser Gln Leu Phe Asn Tyr Phe Arg 115 120 125 Asp Lys Ala Phe Pro Asn Leu Ser Ala Arg Gln Leu Gly Val Met Pro 130 135 140 Asp Leu Val Leu Asp Met Phe Ile Asn Gly Tyr Tyr Leu Asn Val Phe 145 150 155 160 Lys Thr Gln Ser Thr Asp Val Asn Arg Pro Tyr Gln Asp Lys Asp Lys 165 170 175 Arg Gly Gly Ile Phe Asp Ala Val Phe Thr Arg Gly Asn Gln Thr Thr 180 185 190 Leu Leu Thr Ala Arg His Asp Leu Lys Asn Lys Gly Leu Asn Asp Ile 195 200 205 Ser Thr Ile Ile Lys Gln Glu Leu Thr Glu Gly Arg Ala Leu Ala Leu 210 215 220 Ser His Thr Tyr Ala Asn Val Ser Ile Ser His Val Ile Asn Leu Trp 225 230 235 240 Gly Ala Asp Phe Asn Ala Glu Gly Asn Leu Glu Ala Thr Tyr Val Thr 245 250 255 Asp Ser Asp Ala Asn Ala Ser Ile Gly Met Lys Lys Tyr Phe Val Gly 260 265 270 Ile Asn Ala His Gly His Val Ala Ile Ser Ala Lys Lys Ile Glu Gly 275 280 285 Glu Asn Ile Gly Ala Gln Val Leu Gly Leu Phe Thr Leu Ser Ser Gly 290 295 300 Lys Asp Ile Trp Gln Lys Leu Ser 305 310 <210> 46 <211> 292 <212> PRT <213> Artificial Sequence <220> <223> IgG degrading enzyme amino acid sequence <400> 46 Ser Val Trp Thr Lys Gly Val Thr Pro Pro Glu Gln Phe Thr Gln Gly 1 5 10 15 Glu Asp Val Ile His Ala Pro Tyr Leu Ala His Gln Gly Trp Tyr Asp 20 25 30 Ile Thr Lys Ala Phe Asp Gly Ala Asp Asn Leu Leu Cys Gly Ala Ala 35 40 45 Thr Ala Gly Asn Met Leu His Trp Trp Phe Asp Gln Asn Lys Thr Glu 50 55 60 Ile Glu Ala Tyr Leu Ser Lys His Pro Glu Lys Gln Lys Ile Ile Phe 65 70 75 80 Arg Asn Gln Glu Leu Phe Asp Leu Lys Ala Ala Ile Asp Thr Lys Asp 85 90 95 Ser Gln Thr Asn Ser Gln Leu Phe Asn Tyr Phe Arg Asp Lys Ala Phe 100 105 110 Pro Asn Leu Ser Ala Arg Gln Leu Gly Val Met Pro Asp Leu Val Leu 115 120 125 Asp Met Phe Ile Asn Gly Tyr Tyr Leu Asn Val Phe Lys Thr Gln Ser 130 135 140 Thr Asp Val Asn Arg Pro Tyr Gln Asp Lys Asp Lys Arg Gly Gly Ile 145 150 155 160 Phe Asp Ala Val Phe Thr Arg Gly Asn Gln Thr Thr Leu Leu Thr Ala 165 170 175 Arg His Asp Leu Lys Asn Lys Gly Leu Asn Asp Ile Ser Thr Ile Ile 180 185 190 Lys Gln Glu Leu Thr Glu Gly Arg Ala Leu Ala Leu Ser His Thr Tyr 195 200 205 Ala Asn Val Ser Ile Ser His Val Ile Asn Leu Trp Gly Ala Asp Phe 210 215 220 Asn Ala Glu Gly Asn Leu Glu Ala Thr Tyr Val Thr Asp Ser Asp Ala 225 230 235 240 Asn Ala Ser Ile Gly Met Lys Lys Tyr Phe Val Gly Ile Asn Ala His 245 250 255 Gly His Val Ala Ile Ser Ala Lys Lys Ile Glu Gly Glu Asn Ile Gly 260 265 270 Ala Gln Val Leu Gly Leu Phe Thr Leu Ser Ser Gly Lys Asp Ile Trp 275 280 285 Gln Lys Leu Ser 290 <210> 47 <211> 312 <212> PRT <213> Artificial Sequence <220> <223> IgG degrading enzyme amino acid sequence <400> 47 Asp Asp Tyr Gln Arg Asn Ala Thr Glu Ala Tyr Ala Lys Glu Val Pro 1 5 10 15 His Gln Ile Thr Ser Val Trp Thr Lys Gly Val Thr Pro Glu Gln 20 25 30 Phe Thr Gln Gly Glu Asp Val Ile His Ala Pro Tyr Leu Ala His Gln 35 40 45 Gly Trp Tyr Asp Ile Thr Lys Ala Phe Asp Gly Lys Asp Asn Leu Leu 50 55 60 Cys Gly Ala Ala Thr Ala Gly Asn Met Leu His Trp Trp Phe Asp Gln 65 70 75 80 Asn Lys Thr Glu Ile Glu Ala Tyr Leu Ser Lys His Pro Glu Lys Gln 85 90 95 Lys Ile Ile Phe Arg Asn Gln Glu Leu Phe Asp Leu Lys Ala Ala Ile 100 105 110 Asp Thr Lys Asp Ser Gln Thr Asn Ser Gln Leu Phe Asn Tyr Phe Arg 115 120 125 Asp Lys Ala Phe Pro Asn Leu Ser Ala Arg Gln Leu Gly Val Met Pro 130 135 140 Asp Leu Val Leu Asp Met Phe Ile Asn Gly Tyr Tyr Leu Asn Val Phe 145 150 155 160 Lys Thr Gln Ser Thr Asp Val Asn Arg Pro Tyr Gln Asp Lys Asp Lys 165 170 175 Arg Gly Gly Ile Phe Asp Ala Val Phe Thr Arg Gly Asn Gln Thr Thr 180 185 190 Leu Leu Thr Ala Arg His Asp Leu Lys Asn Lys Gly Leu Asn Asp Ile 195 200 205 Ser Thr Ile Ile Lys Gln Glu Leu Thr Glu Gly Arg Ala Leu Ala Leu 210 215 220 Ser His Thr Tyr Ala Asn Val Ser Ile Ser His Val Ile Asn Leu Trp 225 230 235 240 Gly Ala Asp Phe Asn Ala Glu Gly Asn Leu Glu Ala Thr Tyr Val Thr 245 250 255 Asp Ser Asp Ala Asn Ala Ser Ile Gly Met Lys Lys Tyr Phe Val Gly 260 265 270 Ile Asn Ala His Gly His Val Ala Ile Ser Ala Lys Lys Ile Glu Gly 275 280 285 Glu Asn Ile Gly Ala Gln Val Leu Gly Leu Phe Thr Leu Ser Ser Gly 290 295 300 Lys Asp Ile Trp Gln Lys Leu Ser 305 310 <210> 48 <211> 312 <212> PRT <213> Artificial Sequence <220> <223> IgG degrading enzyme amino acid sequence <400> 48 Asp Asp Tyr Gln Arg Asn Ala Thr Glu Ala Tyr Ala Lys Glu Val Pro 1 5 10 15 His Gln Ile Thr Ser Val Trp Thr Lys Gly Val Thr Pro Leu Thr Pro 20 25 30 Glu Gln Phe Thr Gin Gly Glu Asp Val Phe His Ala Pro Tyr Val Ala 35 40 45 Asn Gln Gly Trp Tyr Asp Ile Thr Lys Ala Phe Asp Gly Lys Asp Asn 50 55 60 Leu Leu Cys Gly Ala Ala Thr Ala Gly Asn Met Leu His Trp Trp Phe 65 70 75 80 Asp Gln Asn Lys Asp Gln Ile Lys Arg Tyr Leu Glu Glu His Pro Glu 85 90 95 Lys Gln Lys Ile Asn Phe Asn Gly Glu Asn Met Phe Asp Val Lys Lys 100 105 110 Ala Ile Asp Thr Lys Asn His Gln Leu Asp Ser Lys Leu Phe Asn Tyr 115 120 125 Phe Lys Glu Lys Ala Phe Pro Tyr Leu Ser Ala Lys His Leu Gly Val 130 135 140 Phe Pro Asp His Val Ile Asp Met Phe Ile Asn Gly Tyr Arg Leu Ser 145 150 155 160 Leu Thr Asn His Gly Pro Thr Pro Val Lys Glu Gly Ser Lys Asp Pro 165 170 175 Arg Gly Gly Ile Phe Asp Ala Val Phe Thr Arg Gly Asn Gln Ser Lys 180 185 190 Leu Leu Thr Ser Arg His Asp Phe Lys Asn Lys Asn Leu Asn Asp Ile 195 200 205 Ser Thr Ile Ile Lys Gln Glu Leu Thr Lys Gly Lys Ala Leu Gly Leu 210 215 220 Ser His Thr Tyr Ala Asn Val Arg Ile Asn His Val Ile Asn Leu Trp 225 230 235 240 Gly Ala Asp Phe Asn Ala Glu Gly Asn Leu Glu Ala Thr Tyr Val Thr 245 250 255 Asp Ser Asp Ser Asn Ala Ser Ile Gly Met Lys Lys Tyr Phe Val Gly 260 265 270 Val Asn Ala His Gly His Val Ala Ile Ser Ala Lys Lys Ile Glu Gly 275 280 285 Glu Asn Ile Gly Ala Gln Val Leu Gly Leu Phe Thr Leu Ser Thr Gly 290 295 300 Gln Asp Ser Trp Gln Lys Leu Ser 305 310 <210> 49 <211> 312 <212> PRT <213> Artificial Sequence <220> <223> IgG degrading enzyme amino acid sequence <400> 49 Asp Asp Tyr Gln Arg Asn Ala Thr Glu Ala Tyr Ala Lys Glu Val Pro 1 5 10 15 His Gln Ile Thr Ser Val Trp Thr Lys Gly Val Thr Pro Leu Thr Pro 20 25 30 Glu Gln Phe Thr Gin Gly Glu Asp Val Phe His Ala Pro Tyr Val Ala 35 40 45 Asn Gln Gly Trp Tyr Asp Ile Thr Lys Ala Phe Asp Gly Lys Asp Asn 50 55 60 Leu Leu Cys Gly Ala Ala Thr Ala Gly Asn Met Leu His Trp Trp Phe 65 70 75 80 Asp Gln Asn Lys Asp Gln Ile Lys Arg Tyr Leu Glu Glu His Pro Glu 85 90 95 Lys Gln Lys Ile Asn Phe Arg Gly Glu Asn Met Phe Asp Val Lys Glu 100 105 110 Ala Ile Arg Thr Lys Asn His Gln Leu Asp Ser Lys Leu Phe Glu Tyr 115 120 125 Phe Lys Glu Lys Ala Phe Pro Tyr Leu Ser Ala Lys His Leu Gly Val 130 135 140 Phe Pro Asp His Val Ile Asp Met Phe Ile Asn Gly Tyr Arg Leu Ser 145 150 155 160 Leu Thr Asn His Gly Pro Thr Pro Val Lys Lys Gly Ser Lys Asp Pro 165 170 175 Arg Gly Gly Ile Phe Asp Ala Val Phe Thr Arg Gly Asn Gln Ser Lys 180 185 190 Leu Leu Thr Ser Arg His Asp Phe Lys Asn Lys Asn Leu Asn Asp Ile 195 200 205 Ser Thr Ile Ile Lys Ser Glu Leu Thr Asn Gly Lys Ala Leu Gly Leu 210 215 220 Ser His Thr Tyr Ala Asn Val Arg Ile Asn His Val Ile Asn Leu Trp 225 230 235 240 Gly Ala Asp Phe Asn Ala Glu Gly Asn Leu Glu Ala Thr Tyr Val Thr 245 250 255 Asp Ser Asp Ser Asn Ala Ser Ile Gly Met Lys Lys Tyr Phe Val Gly 260 265 270 Val Asn Lys His Gly His Val Ala Ile Ser Ala Lys Lys Ile Glu Gly 275 280 285 Glu Asn Ile Gly Ala Gln Val Leu Gly Leu Phe Thr Leu Ser Thr Gly 290 295 300 Gln Asp Ser Trp Gln Lys Leu Asn 305 310 <210> 50 <211> 312 <212> PRT <213> Artificial Sequence <220> <223> IgG degrading enzyme amino acid sequence <400> 50 Asp Asp Tyr Gln Arg Asn Ala Thr Glu Ala Tyr Ala Lys Glu Val Pro 1 5 10 15 His Gln Ile Thr Ser Val Trp Thr Lys Gly Val Thr Pro Leu Thr Pro 20 25 30 Glu Gln Phe Thr Gin Gly Glu Asp Val Phe His Ala Pro Tyr Val Ala 35 40 45 Asn Gln Gly Trp Tyr Asp Ile Thr Lys Thr Phe Asn Gly Lys Asp Asp 50 55 60 Leu Leu Cys Gly Ala Ala Thr Ala Gly Asn Met Leu His Trp Trp Phe 65 70 75 80 Asp Gln Asn Lys Asp Gln Ile Lys Arg Tyr Leu Glu Glu His Pro Glu 85 90 95 Lys Gln Lys Ile Asn Phe Asn Gly Glu Gln Met Phe Asp Val Lys Glu 100 105 110 Ala Ile Asp Thr Lys Asn His Gln Leu Asp Ser Lys Leu Phe Glu Tyr 115 120 125 Phe Lys Glu Lys Ala Phe Pro Tyr Leu Ser Thr Lys His Leu Gly Val 130 135 140 Phe Pro Asp His Val Ile Asp Met Phe Ile Asn Gly Tyr Arg Leu Ser 145 150 155 160 Leu Thr Asn His Gly Pro Thr Pro Val Lys Glu Gly Ser Lys Asp Pro 165 170 175 Arg Gly Gly Ile Phe Asp Ala Val Phe Thr Arg Gly Asn Gln Ser Lys 180 185 190 Leu Leu Thr Ser Arg His Asp Phe Lys Glu Lys Asn Leu Lys Glu Ile 195 200 205 Ser Asp Leu Ile Lys Gln Glu Leu Thr Glu Gly Lys Ala Leu Gly Leu 210 215 220 Ser His Thr Tyr Ala Asn Val Arg Ile Asn His Val Ile Asn Leu Trp 225 230 235 240 Gly Ala Asp Phe Asp Ala Glu Gly Asn Leu Lys Ala Ile Tyr Val Thr 245 250 255 Asp Ser Asp Ser Asn Ala Ser Ile Gly Met Lys Lys Tyr Phe Val Gly 260 265 270 Val Asn Ala Ala Gly Lys Val Ala Ile Ser Ala Lys Lys Ile Glu Gly 275 280 285 Glu Asn Ile Gly Ala Gln Val Leu Gly Leu Phe Thr Leu Ser Thr Gly 290 295 300 Gln Asp Ser Trp Asn Gln Thr Ser 305 310 <210> 51 <211> 312 <212> PRT <213> Artificial Sequence <220> <223> IgG degrading enzyme amino acid sequence <400> 51 Asp Asp Tyr Gln Arg Asn Ala Thr Glu Ala Tyr Ala Lys Glu Val Pro 1 5 10 15 His Gln Ile Thr Ser Val Trp Thr Lys Gly Val Thr Pro Leu Thr Pro 20 25 30 Glu Gln Phe Thr Gin Gly Glu Asp Val Phe His Ala Pro Tyr Val Ala 35 40 45 Asn Gln Gly Trp Tyr Asp Ile Thr Lys Thr Phe Asn Gly Lys Asp Asp 50 55 60 Leu Leu Cys Gly Ala Ala Thr Ala Gly Asn Met Leu His Trp Trp Phe 65 70 75 80 Asp Gln Asn Lys Asp Gln Ile Lys Arg Tyr Leu Glu Glu His Pro Glu 85 90 95 Lys Gln Lys Ile Asn Phe Arg Gly Glu Gln Met Phe Asp Val Lys Glu 100 105 110 Ala Ile Arg Thr Lys Asn His Gln Leu Asp Ser Lys Leu Phe Glu Tyr 115 120 125 Phe Lys Glu Lys Ala Phe Pro Tyr Leu Ser Thr Lys His Leu Gly Val 130 135 140 Phe Pro Asp His Val Ile Asp Met Phe Ile Asn Gly Tyr Arg Leu Ser 145 150 155 160 Leu Thr Asn His Gly Pro Thr Pro Val Lys Lys Gly Ser Lys Asp Pro 165 170 175 Arg Gly Gly Ile Phe Asp Ala Val Phe Thr Arg Gly Asn Gln Ser Lys 180 185 190 Leu Leu Thr Ser Arg His Asp Phe Lys Glu Lys Asn Leu Lys Glu Ile 195 200 205 Ser Asp Leu Ile Lys Glu Glu Leu Thr Lys Gly Lys Ala Leu Gly Leu 210 215 220 Ser His Thr Tyr Ala Asn Val Arg Ile Asn His Val Ile Asn Leu Trp 225 230 235 240 Gly Ala Asp Phe Asp Ala Glu Gly Asn Leu Lys Ala Ile Tyr Val Thr 245 250 255 Asp Ser Asp Ser Asn Ala Ser Ile Gly Met Lys Lys Tyr Phe Val Gly 260 265 270 Val Asn Lys Ala Gly Lys Val Ala Ile Ser Ala Lys Lys Ile Glu Gly 275 280 285 Glu Asn Ile Gly Ala Gln Val Leu Gly Leu Phe Thr Leu Ser Thr Gly 290 295 300 Gln Asp Ser Trp Asn Gln Thr Asn 305 310 <210> 52 <211> 310 <212> PRT <213> Artificial Sequence <220> <223> IgG degrading enzyme amino acid sequence <400> 52 Asp Asp Tyr Gln Arg Asn Ala Thr Glu Ala Tyr Ala Lys Glu Val Pro 1 5 10 15 His Gln Ile Thr Ser Val Trp Thr Lys Gly Val Thr Pro Glu Gln 20 25 30 Phe Thr Gln Gly Glu Asp Val Ile His Ala Pro Tyr Val Ala Asn Gln 35 40 45 Gly Trp Tyr Asp Ile Thr Lys Ala Phe Asp Gly Lys Asp Asn Leu Leu 50 55 60 Cys Gly Ala Ala Thr Ala Gly Asn Met Leu His Trp Trp Phe Asp Gln 65 70 75 80 Asn Lys Asp Gln Ile Lys Arg Tyr Leu Glu Glu His Pro Glu Lys Gln 85 90 95 Lys Ile Asn Phe Arg Gly Glu Gln Met Phe Asp Val Lys Lys Ala Ile 100 105 110 Asp Thr Lys Asn His Gln Leu Asp Ser Lys Leu Phe Asn Tyr Phe Lys 115 120 125 Glu Lys Ala Phe Pro Gly Leu Ser Ala Arg Arg Ile Gly Val Phe Pro 130 135 140 Asp His Val Ile Asp Met Phe Ile Asn Gly Tyr Arg Leu Ser Leu Thr 145 150 155 160 Asn His Gly Pro Thr Pro Val Lys Glu Gly Ser Lys Asp Pro Arg Gly 165 170 175 Gly Ile Phe Asp Ala Val Phe Thr Arg Gly Asn Gln Ser Lys Leu Leu 180 185 190 Thr Ser Arg His Asp Phe Lys Asn Lys Asn Leu Asn Asp Ile Ser Thr 195 200 205 Ile Ile Lys Gln Glu Leu Thr Lys Gly Lys Ala Leu Gly Leu Ser His 210 215 220 Thr Tyr Ala Asn Val Ser Ile Asn His Val Ile Asn Leu Trp Gly Ala 225 230 235 240 Asp Phe Asn Ala Glu Gly Asn Leu Glu Ala Thr Tyr Val Thr Asp Ser 245 250 255 Asp Ser Asn Ala Ser Ile Gly Met Lys Lys Tyr Phe Val Gly Val Asn 260 265 270 Ala His Gly His Val Ala Ile Ser Ala Lys Lys Ile Glu Gly Glu Asn 275 280 285 Ile Gly Ala Gln Val Leu Gly Leu Phe Thr Leu Ser Thr Gly Gln Asp 290 295 300 Ser Trp Gln Lys Leu Ser 305 310

Claims (124)

A method for reducing the content of neutralizing antibody against a recombinant adeno-associated virus (AAV) vector in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a composition that promotes degradation of the neutralizing antibody, Way. The method of claim 1 , wherein the neutralizing antibody is IgG, IgM, IgE or IgA. The method of claim 2 , wherein the neutralizing antibody is IgG. 4. The method according to any one of claims 1 to 3, wherein said recombinant AAV vector is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh8, AAVrh10, AAVrh32.33 , AAVrh74, avian AAV or bovine AAV vector. 5. The method of claim 4, wherein the AAV vector is a wild-type AAV vector. 5. The method of claim 4, wherein the AAV vector is a mutant AAV vector. 7. The method of any one of claims 1-6, wherein the recombinant AAV vector comprises a heterologous nucleic acid encoding a therapeutic protein or therapeutic RNA. 8. The method of any one of claims 1 to 7, wherein after administration of the composition, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70% of the antibody , wherein at least 80%, at least 90% or at least 99% are degraded in the subject. 9. The method of any one of claims 1-8, wherein the composition comprises an antibody-degrading enzyme or fragment thereof. 9. The method of any one of claims 1-8, wherein the composition comprises a vector comprising a polynucleotide encoding an antibody-degrading enzyme or fragment thereof. 11. The method of claim 9 or 10, wherein the antibody-degrading enzyme or fragment thereof has cysteine protease activity. 12. The method of any one of claims 9-11, wherein the antibody-degrading enzyme specifically cleaves IgG. 13. The method according to any one of claims 9 to 12, wherein the antibody-degrading enzyme or fragment thereof is derived from the genus Streptococcus. 14. The method of any one of claims 9-13, wherein the antibody-degrading enzyme comprises an amino acid sequence that is at least 90% or at least 95% identical to the amino acid sequence of SEQ ID NO: 1. 15. The method of claim 14, wherein the antibody-degrading enzyme comprises the amino acid sequence of SEQ ID NO: 1. 16. The method of any one of claims 1-15, wherein the composition comprises a fusion protein comprising a first protein and a second protein, wherein the first protein is an antibody-degrading enzyme or fragment thereof. The method of claim 16 , wherein the first protein and the second protein are separated by a linker. 18. The method of claim 16 or 17, wherein the second protein is an IgG protease. 16. The method of any one of claims 9-15, wherein the subject is administered from about 0.1 mg/kg to about 100 mg/kg of the antibody-degrading enzyme or fragment thereof. 20. The method of any one of claims 1-19, wherein said administering reduces binding of said antibody to an Fc receptor. 21. The method of any one of claims 1-20, wherein the composition is administered intravenously. 22. The method of any one of claims 1-21, wherein the composition comprises a pharmaceutically acceptable carrier and/or diluent. 23. The method of any one of claims 1-22, wherein the subject is a human. 24. The method of any one of claims 1-23, wherein the subject is treated with the recombinant adeno-associated virus (AAV) vector prior to administration of the composition. 24. The composition of any one of claims 1-23, wherein the subject is not treated with the recombinant AAV prior to administration of the composition. A method of preparing a subject for treatment with a recombinant adeno-associated virus (AAV) vector, wherein the method (a) promotes degradation of neutralizing antibodies to the AAV vector, and/or (b) neutralizes to Fc receptors. A method comprising administering to the subject an effective amount of a composition that reduces binding of an antibody. The method of claim 26 , wherein the neutralizing antibody is IgG, IgM, IgE or IgA. The method of claim 27 , wherein the neutralizing antibody is IgG. 29. The method of any one of claims 26-28, wherein said recombinant AAV vector is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh8, AAVrh10, AAVrh32.33. , AAVrh74, avian AAV or bovine AAV vector. 30. The method of claim 29, wherein the AAV vector is a wild-type AAV vector. 30. The method of claim 29, wherein the AAV vector is a mutant AAV vector. 32. The method of any one of claims 26-31, wherein the recombinant AAV comprises a heterologous nucleic acid encoding a therapeutic protein or therapeutic RNA. 33. The method of any one of claims 26-32, wherein at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 99% is degraded in the body of the subject. 34. The method of any one of claims 26-33, wherein the composition comprises an antibody-degrading enzyme or fragment thereof. 34. The method of any one of claims 26-33, wherein the composition comprises a vector comprising a polynucleotide encoding an antibody-degrading enzyme or fragment thereof. 36. The method of claim 34 or 35, wherein the antibody-degrading enzyme or fragment thereof has cysteine protease activity. 37. The method of any one of claims 26-36, wherein the antibody-degrading enzyme specifically cleaves IgG. 38. The method according to any one of claims 26 to 37, wherein the antibody-degrading enzyme or fragment thereof is from the genus Streptococcus . 39. The method of any one of claims 26-38, wherein the antibody-degrading enzyme comprises an amino acid sequence that is at least 90% or at least 95% identical to the amino acid sequence of SEQ ID NO: 1. 40. The method of claim 39, wherein the antibody-degrading enzyme comprises the amino acid sequence of SEQ ID NO:1. 41. The method of any one of claims 26-40, wherein the composition comprises a fusion protein comprising a first protein and a second protein, wherein the first protein is an antibody-degrading enzyme or fragment thereof. 42. The method of claim 41, wherein the first protein and the second protein are separated by a linker. 43. The method of claim 41 or 42, wherein the second protein is an IgG protease. 44. The method of any one of claims 34-43, wherein the subject is administered from about 0.1 mg/kg to about 100 mg/kg of the antibody-degrading enzyme or fragment thereof. 45. The method of any one of claims 28-44, wherein said administering reduces binding of the antibody to an Fc receptor. 46. The method of any one of claims 26-45, wherein the composition is administered intravenously. 47. The method of any one of claims 26-46, wherein the composition comprises a pharmaceutically acceptable carrier and/or diluent. 48. The method of any one of claims 26-47, wherein the subject is a human. A method of treating a recombinant adeno-associated virus (AAV) vector in a subject in need, said method comprising:
(i) administering to the subject an effective amount of a composition that (a) promotes degradation of the neutralizing antibody to an AAV vector, and/or (b) reduces binding of the neutralizing antibody to an Fc receptor; and
(ii) administering to said subject an effective amount of said AAV vector;
A method comprising 50. The method of claim 49, wherein the AAV vector is administered concurrently with the composition. 50. The method of claim 49, wherein the AAV vector is administered after administration of the composition. 50. The method of claim 49, wherein the AAV vector is administered prior to administering the composition. 53. The method of any one of claims 49-52, wherein the method further comprises administering to the subject a second AAV vector. 54. The method of claim 53, wherein the AAV vector and the second AAV vector comprise an AAV capsid protein of the same serotype. 54. The method of claim 53, wherein the AAV vector and the second AAV vector comprise AAV capsid proteins of different serotypes. 56. The method of any one of claims 45-55, wherein the neutralizing antibody is IgG, IgM, IgE or IgA. 57. The method of claim 56, wherein the neutralizing antibody is an IgG. 58. The method of any one of claims 49-57, wherein said recombinant AAV vector is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh8, AAVrh10, AAVrh32.33. , AAVrh74, avian AAV or bovine AAV vector. 59. The method of claim 58, wherein the AAV vector is a wild-type AAV vector. 59. The method of claim 58, wherein the AAV vector is a mutant AAV vector. 61. The method of any one of claims 49-60, wherein the recombinant AAV comprises a heterologous nucleic acid encoding a therapeutic protein or therapeutic RNA. 62. The method of any one of claims 49-61, wherein after administration of the composition, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70% of the antibody , at least 80%, at least 90% or at least 99% is degraded. 63. The method of any one of claims 49-62, wherein the composition comprises an antibody-degrading enzyme or fragment thereof. 63. The method of any one of claims 49-62, wherein the composition comprises a vector comprising a polynucleotide encoding an antibody-degrading enzyme or fragment thereof. 65. The method of claim 63 or 64, wherein the antibody-degrading enzyme or fragment thereof has cysteine protease activity. 66. The method of any one of claims 63-65, wherein the antibody-degrading enzyme specifically cleaves IgG. 67. The method according to any one of claims 63 to 66, wherein the antibody-degrading enzyme or fragment thereof is from the genus Streptococcus. 68. The method of any one of claims 63-67, wherein the antibody-degrading enzyme comprises an amino acid sequence that is at least 90% or at least 95% identical to the amino acid sequence of SEQ ID NO: 1. 69. The method of claim 68, wherein the antibody-degrading enzyme comprises the amino acid sequence of SEQ ID NO: 1. 70. The method of any one of claims 49-69, wherein the composition comprises a fusion protein comprising a first protein and a second protein, wherein the first protein is an antibody-degrading enzyme or fragment thereof. 71. The method of claim 70, wherein the first protein and the second protein are separated by a linker. 72. The method of claim 70 or 71, wherein the second protein is an IgG protease. 73. The method of any one of claims 63-72, wherein the subject is administered from about 0.1 mg/kg to about 100 mg/kg of the antibody-degrading enzyme or fragment thereof. 74. The method of any one of claims 49-73, wherein said administering reduces binding of said antibody to an Fc receptor. 75. The method of any one of claims 49-74, wherein the composition is administered intravenously. 76. The method of any one of claims 49-75, wherein the composition comprises a pharmaceutically acceptable carrier and/or diluent. 77. The method of any one of claims 49-76, wherein the subject is a human. A method of treating a second recombinant adeno-associated virus (AAV) vector in a subject in need thereof previously treated with a first recombinant adeno-associated virus (AAV) vector, said method comprising:
(i) an effective amount of a composition that (a) promotes degradation of the neutralizing antibody to the first and/or second recombinant AAV vector, and/or (b) reduces binding of the neutralizing antibody to an Fc receptor in the subject; administering; and
(ii) administering to said subject an effective amount of a second recombinant AAV vector;
A method comprising 79. The method of claim 78, wherein the first recombinant AAV and the second recombinant AAV are of the same serotype. 79. The method of claim 78, wherein the first recombinant AAV and the second recombinant AAV are different serotypes. 81. The method of any one of claims 78-80, wherein the neutralizing antibody is IgG, IgM, IgE or IgA. 82. The method of claim 81, wherein the neutralizing antibody is IgG. 83. The method of any one of claims 76-82, wherein said recombinant AAV vector is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh8, AAVrh10, AAVrh32.33. , AAVrh74, avian AAV or bovine AAV vector. 84. The method of claim 83, wherein the AAV vector is a wild-type AAV vector. 84. The method of claim 83, wherein the AAV vector is a mutant AAV vector. 86. The method of any one of claims 78-85, wherein the recombinant AAV comprises a heterologous nucleic acid encoding a therapeutic protein or therapeutic RNA. 87. The method of any one of claims 78-86, wherein at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, of the antibody after administration of the composition, at least 80%, at least 90% or at least 99% is degraded. 88. The method of any one of claims 78-87, wherein the composition comprises an antibody-degrading enzyme or fragment thereof. 88. The method of any one of claims 78-87, wherein the composition comprises a vector comprising a polynucleotide encoding an antibody-degrading enzyme or fragment thereof. 90. The method of claim 88 or 89, wherein the antibody-degrading enzyme or fragment thereof has cysteine protease activity. 91. The method of any one of claims 78-90, wherein the antibody-degrading enzyme specifically cleaves IgG. 92. The method of any one of claims 78-91, wherein the antibody-degrading enzyme or fragment thereof is from the genus Streptococcus . 93. The method of any one of claims 78-92, wherein the antibody-degrading enzyme comprises an amino acid sequence that is at least 90% or at least 95% identical to the amino acid sequence of SEQ ID NO: 1. 94. The method of claim 93, wherein the antibody-degrading enzyme comprises the amino acid sequence of SEQ ID NO: 1. 95. The method of any one of claims 87-94, wherein the composition comprises a fusion protein comprising a first protein and a second protein, wherein the first protein is an antibody-degrading enzyme or fragment thereof. 96. The method of claim 95, wherein the first protein and the second protein are separated by a linker. 97. The method of claim 95 or 96, wherein the second protein is an IgG protease. 98. The method of any one of claims 78-97, wherein the subject is administered from about 0.1 mg/kg to about 100 mg/kg of the antibody-degrading enzyme or fragment thereof. 99. The method of any one of claims 78-98, wherein said administering reduces binding of said antibody to an Fc receptor. 101. The method of any one of claims 78-99, wherein the composition is administered intravenously. 101. The method of any one of claims 78-100, wherein the composition comprises a pharmaceutically acceptable carrier and/or diluent. 102. The method of any one of claims 78-101, wherein the subject is a human. A method of reducing neutralizing antibodies to an adeno-associated virus (AAV) vector comprising a heterologous nucleic acid in a subject in need thereof, comprising: in said subject an effective amount of an AAV vector, and (a) an antibody to the AAV vector or a heterologous nucleic acid; A method comprising administering a composition that promotes degradation of the encoded recombinant protein and/or (b) decreases binding of the antibody to an Fc receptor. 104. The method of claim 103, wherein the antibody is an IgG. 105. The method of claim 103 or 104, wherein the AAV vector is administered to the subject concurrently with the composition. 105. The method of claim 103 or 104, wherein the AAV vector is administered after administration of the composition to the subject. 105. The method of claim 103 or 104, wherein the AAV vector is administered prior to administering the composition to the subject. 108. The method of claim 107, further comprising administering at least one second AAV vector comprising a second heterologous nucleic acid. 109. The method of claim 108, wherein the AAV vector and the second AAV vector comprise an AAV capsid protein of the same serotype. 109. The method of claim 108, wherein the AAV vector and the second AAV vector comprise AAV capsid proteins of different serotypes. 112. The method of any one of claims 102-110, wherein the composition further comprises a pharmaceutically acceptable carrier and/or diluent. 112. The method of any one of claims 102-111, wherein the composition promotes degradation of the antibody. 113. The method of claim 112, wherein the level of said antibody in said subject is reduced to a level in the range of about 95% to about 0.01% relative to the level of antibody in a control subject, wherein said control subject is administered an AAV vector but not administered said composition. Not an object, a method. 114. The method of claim 112 or 113, wherein the composition comprises an antibody-degrading enzyme or fragment thereof. 114. The method of claim 112 or 113, wherein the composition comprises a vector comprising a polynucleotide encoding an antibody-degrading enzyme or fragment thereof. 116. The method of claim 114 or 115, wherein the antibody-degrading enzyme or fragment thereof comprises an IgG cysteine protease activity. 117. The method of any one of claims 114-116, wherein the antibody-degrading enzyme or fragment thereof is from the genus Streptococcus . 118. The method of any one of claims 114-117, wherein the antibody-degrading enzyme comprises an amino acid sequence that is at least 50% identical to the amino acid sequence of SEQ ID NO: 1. 119. The method of any one of claims 114-118, wherein the antibody-degrading enzyme comprises the amino acid sequence of SEQ ID NO:1. 120. The method of any one of claims 114 to 119, wherein the composition comprises an antibody-degrading enzyme or fragment thereof; and a fusion protein comprising a second protein. 121. The method of claim 120, wherein the second protein is an IgG protease. 122. The method of any one of claims 114-121, wherein the subject is administered with an antibody-degrading enzyme or fragment thereof from about 0.1 mg/kg to about 100 mg/kg. 123. The method of any one of claims 108-122, wherein the subject is a human. 103. The method of claim 102, wherein the composition reduces binding of the antibody to an Fc receptor.

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